Gotowa bibliografia na temat „P53-regulated genes”

Identification of p73 as a structural homolog of p53 fueled early studies aimed at determining if it was capable of performing p53-like functions. This led to a conundrum as p73 was discovered to be hardly mutated in cancers, and yet, TAp73, the full-length form, was found capable of performing p53-like functions, including transactivation of many p53 target genes in cancer cell lines. Generation of mice lacking p73/TAp73 revealed a plethora of developmental defects, with very limited spontaneous tumors arising only at a later stage. Concurrently, novel TAp73 target genes involved in cellular growth promotion that are not regulated by p53 were identified, mooting the possibility that TAp73 may have diametrically opposite functions to p53 in tumorigenesis. We have therefore comprehensively evaluated the TAp73 target genes identified and validated in human cancer cell lines, to examine their contextual relevance. Data from focused studies aimed at appraising if p53 targets are also regulated by TAp73—often by TAp73 overexpression in cell lines with non-functional p53—were affirmative. However, genome-wide and phenotype-based studies led to the identification of TAp73-regulated genes involved in cellular survival and thus, tumor promotion. Our analyses therefore suggest that TAp73 may not necessarily be p53’s natural substitute in enforcing tumor suppression. It has likely evolved to perform unique functions in regulating developmental processes and promoting cellular growth through entirely different sets of target genes that are not common to, and cannot be substituted by p53. The p53-related targets initially reported to be regulated by TAp73 may therefore represent an experimental possibility rather than the reality.

Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by γ radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer’s disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer’s disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 µg/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 µg/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-?B were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-?B was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-?B are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Physical Sciences<br>Faculty of Science, Environment, Engineering and Technology<br>Full Text

The breast tumor suppressor hCLCA2 is a putative chloride regulator that is expressed in normal breast epithelial cells and frequently down-regulated in breast cancers. The first CLCA protein was described as a calcium-activated, plasma-membrane chloride channel having four or five transmembrane pass structure that could form a channel pore. However, CLCA topology is inconsistent with chloride channel function. We showed that hCLCA2 itself is unlikely to form a channel as it has only a single transmembrane segment with a short cytoplasmic tail and is mostly extracellular. Moreover, the N-terminal 109-kDa ectodomain is cleaved at the cell surface and shed into the medium while the 35-kDa C-terminal product is retained by the cell membrane. The general goal of my project was to study the function of this novel protein and its role in breast cancer. In addition to its role in chloride regulation, hCLCA2 behaves as a tumor suppressor gene that is frequently down-regulated in breast cancer. We previously demonstrated that murine homologs of hCLCA2 are transcriptionally induced during mammary involution, when the gland shuts down and 80% of the mammary epithelial cells die by apoptosis. In cell culture, conditions that cause G1 arrest such as contact inhibition and depriving cells of growth factors and anchorage induced these genes. Therefore, one of the goals of this project was to find if this is true of hCLCA2 in human breast epithelial cells. We found that hCLCA2 was induced by the above mentioned stresses and by pharmacological blockage of cell survival signaling. In addition, we found that DNA-damaging agents doxorubicin and aphidicolin potently induced hCLCA2 in p53-positive cell lines such as MCF-7 but not in p53-deficient cells such as MDA-MB231. An adenovirus encoding p53 induced hCLCA2 expression in a broad spectrum of breast cancer cell lines while a control virus did not, suggesting that hCLCA2 is a p53-inducible gene. To further test the hypothesis, we performed chromatin immunoprecipitation (ChIP) to determine whether p53 bound to the hCLCA2 promoter. This analysis showed that p53 binds directly to the hCLCA2 promoter between -157 and -359bp upstream of the translation initiation site. This segment was required for the p53-dependent expression of an hCLCA2-luciferase fusion gene. Point mutation of the p53 consensus binding motif abolished this induction. Induction of hCLCA2 in MCF-7 cells by doxorubicin was inhibited by p53 knockdown and by p53 inhibitor pifithrin, indicating that p53 activates the endogenous hCLCA2 promoter in response to DNA damage. An adenovirus encoding hCLCA2 induced a cell cycle lag in G0/G1 phase, decreased intracellular pH from 7.49 to 6.7, caused Bax and Bad translocation to the mitochondria, activated caspases, induced PARP cleavage, and promoted apoptosis. Conversely, hCLCA2 knockdown enhanced proliferation of epithelial MCF10A cells and reduced sensitivity to doxorubicin. These results reveal the molecular mechanism of hCLCA2 induction and downstream events that may provide protection from tumorigenesis. Epithelial cells acquire mesenchymal characteristics by undergoing phenotypic and genotypic changes during cancer progression. An early step in the epithelial to mesenchymal transition (EMT) is the disruption of intercellular connections due to loss of epithelial cadherins. We find that expression of tumor suppressor hCLCA2 is strongly associated with epithelial differentiation and that induction of EMT by mesenchymal transcription factors represses its expression. Moreover, we found that knockdown of hCLCA2 by RNA interference results in disruption of cell-cell junctions by downregulating E-cadherin. This also imparts invasiveness and anoikis-resistance to epithelial cells but is insufficient to induce full EMT. However, activation of Ras oncogene in combination with hCLCA2 knockdown is sufficient to induce full EMT in vitro. These findings indicate that, like E-cadherin, hCLCA2 is required for epithelial differentiation and that its loss during tumor progression may contribute to metastasis.

DNA damage transactivates TP53-regulated surveillance mechanisms that are crucial in maintaining cellular integrity and suppressing tumorigenesis. TP53 mediates this directly by transcriptionally modulating gene and microRNA (miRNA) expression and by regulating miRNA biogenesis through interaction with the DROSHA complex. However, the regulative mechanism of miRNA-AGO2 loading and the global change in AGO2 binding to its gene targets in response to DNA damage have not been investigated yet. In addition, the role of other non-coding RNAs, such as snoRNAs, in the TP53-mediated response to DNA damage has not yet been defined. Here we identify a novel group of TP53-regulated miRNAs and show that DNA damage induces and reduces the loading of a subset of miRNAs, including the let-7 family members onto AGO2, in a TP53-dependent manner and that this previously undescribed process is most likely the result of TP53 binding to AGO2. These findings indicate that TP53 control of AGO2 loading is a new mechanism of miRNA regulation in carcinogenesis. Using AGO2 RIP-Seq and PAR-CLIP we also show that TP53 modulates the reduction, induction and remodelling of AGO2 binding to the 3'UTR of different mRNA targets at specific RNA motifs. Furthermore, we determine on a transcriptome-wide level the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence or absence of TP53. We also show that those miRNAs whose cellular abundance or differential loading onto AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits that fine tune gene expression levels in response to DNA damage to permit DNA repair or the initiation of programmed cell death. Finally, we demonstrate a relationship between TP53 and the GAS5-derived snoRNAs both in cancer cell lines and human tissue samples which implies that this class of non-coding RNAs might also be involved in coordinating the TP53-mediated response. These findings provide a novel insight into the complexities surrounding the role of non-coding RNAs in the TP53 response to DNA damage and their relevance to carcinogenesis.

碩士<br>國立陽明大學<br>生物化學研究所<br>93<br>p53 tumor suppressor is the most frequently mutated gene in human cancers. p53 must achieve its role in tumor suppression through transactivation of target genes, i.e. genes involved in cell survival or apoptosis regulation. Aiming further understanding of p53, we set out to search and to identify novel p53 downstream targets. By comparing gene expression patterns under basal or p53 activation conditions using microarray, we discovered two potential target genes, Rad and PRL-1, whitch can be up-regulated when p53 expression is induced by DNA damage or ectopically. Rad is a small G protein belonging to the RGK family, while PRL-1 is a protein tyrosine phosphatase; both are with unknown function. Rad has a p53 response element (p53RE) in the 5’ promoter region and PRL-1 has a p53RE in intron its 1. Using chromatin immunoprecipitation (ChIP) and luciferase reporter assay, we demonstrate that p53 can bind to p53REs of Rad and PRL-1 and activates their mRNA transcription under adriamycin and UV damage or p53 overexpression. p73, which has sequence hormology to p53 in the DNA binding domain, also transactivates Rad and PRL-1 through p53RE. p73 β and δ confer strongest activation among four p73 isoforms. Previous studies on Rad have revealed that Rad binds Rho-associated kinase ROCK and subsequently inhibits ROCK kinase activity upon MYPT-1 and shortens the length of neurites in neuroblastoma cells. We also show that Rad binds to ROCK I and ROCK II in vivo and repress RhoA-induced NF-κB and cyclin D1 promoter activation equally well as ROCK inhibitor Y27632 does. Rad overexpressed Cos7 shows growth suppression evidenced by colony formation assay. Our results prove that Rad and PRL-1 are p53 targets and increase mRNA levels in a p53-dependent manner. Rad protein levels also increase under p53 activation. p53 binds to promoter and intron region of Rad and PRL-1, respectively, and transactivates Rad and PRL-1 expression. Further experiments could reveal new function of p53 in regulating Rad and PRL-1 in glucose metabolism, NF-κB signaling and mechanism of controlling cell cycle reentry.

博士<br>國立陽明大學<br>生物化學研究所<br>88<br>p53 is a tumor suppressor gene that functions as a guardian to maintain the integrity of the genome. It is the most frequently mutated and disrupted target in human cancers. Activation of p53 triggers cells into cell cycle arrest, apoptosis and differentiation, depending on the cell type, cellular environment and extracellular stimuli. p53 acts through direct interaction with other proteins or as a transcription factor regulating the expression of its downstream effector genes. A key approach to elucidating the biological function of p53 is to look for its direct target genes. By mRNA differential display analysis on murine IW32 erythroleukemia cells containing a temperature sensitive p53 allele (tsp53val-135) cultured at 32.5°C and 38.5°C, two novel p53-regulated genes, designated mDDA1 and mDDA3, have been identified. Induction of mDDA1 and mDDA3 occurred in all IW32 sublines expressing tsp53val-135 cultured at permissive temperature. Elevated levels of mDDA1 and mDDA3 transcripts were detected within 1 h and 2 h, respectively, after down-shifting the temperature from 38.5℃ to 32.5℃. Moreover, actinomycin D, but not cycloheximide, inhibited the p53-dependent induction of these genes, suggesting that their activation was through transcriptional regulation and did not require de novo protein synthesis. DDA1 transcript was predominantly expressed in mouse liver and human skeletal muscle, while that of DDA3 was found in multiple mouse and human tissues. Using 5''-RACE and a PCR-based genome walking method, full-length cDNAs and genomic DNAs of mDDA1 and mDDA3 were cloned. The mDDA1 cDNA encodes a putative protein of 498 amino acids containing 12 transmembrane domains. The genomic DNA of mDDA1 is 18 kb in length, consisting of six exons and five introns. Four putative p53 recognition motifs are found in intron 1; at least one of these sites was demonstrated to support the responsiveness to wild-type, but not mutant p53, in a transient transfection assay. Sequence comparison revealed that mDDA1 shares 73% and 90% identity in its nucleotide and protein sequences, respectively, to the newly identified human thiamine transporter gene (hTHTR-1). The gene structures of mDDA1 and hTHTR-1 are similar; both contain identical numbers of exon and intron, and the RNA splicing joint sites are also conserved. Therefore, mDDA1 is very likely to be the mouse homologue of the hTHTR-1. Based on these analyses, mDDA1 was hereafter named mouse THTR-1 (mTHTR-1). mTHTR-1 also shares 40% identity in its protein sequence to the reduced folate carrier-1 (RFC-1) from human and mouse. However, We have shown that mTHTR-1 exhibited very low methotrexate uptake activity when compared to that of RFC-1. The mDDA3 gene is composed of eight exons and seven introns, and a putative p53 recognition motif was found in its intron 3. Sequence analysis of the cloned mDDA3 cDNAs indicated that there were at least seven types of transcripts, differed only in their 5''-termini. Results from primer extension and RNase protection assays suggest that the 5''-heterogeneity of mDDA3 mRNAs may result from multiple transcriptional initiations as well as alternative splicing of the transcripts. Three of these mDDA3 cDNAs contain uninterrupted open reading frames; two of them encode a protein of 329 amino acids (mDDA3S) and the third, 344 amino acids (mDDA3L). Except for a 15 amino acid-extension at the N-terminus of mDDA3L, the two proteins are identical in sequence. The mDDA3 protein is rich in serine and proline; it contains one coiled-coil domain and six "PXXP" motifs capable of interacting SH3 containing proteins. Full-length human DDA3 cDNA has been obtained by homology searches of a human EST database; sequence analysis indicates that hDDA3 encodes a protein of 333 amino acids that shares 68% identity to mDDA3S. Overexpression of both mTHTR-1 and mDDA3 in H1299 non-small cell lung carcinoma cells partially suppressed colony formation. In summary, we have cloned and characterized two p53 transcriptional target genes mTHTR-1 and DDA3. Our analyses have implicated mTHTR-1 in thiamine homeostasis and suggested a role of DDA3 in the p53-mediated growth suppression. 英文摘要--------------------------------------------------------------------------------------- 3 (壹)‧緒論 1.1 細胞的生長與死亡之調控------------------------------------------------------- 5 1.2 p53 tumor suppressor ------------------------------------------------------------- 7 1.3 p53蛋白的結構與功能----------------------------------------------------------- 7 1.3.1 Transactivation domain ------------------------------------------------------- 7 1.3.2 Proline-rich domain ------------------------------------------------------------ 8 1.3.3 Central DNA-binding core domain ----------------------------------------- 8 1.3.4 C端oligomerization domain -------------------------------------------------- 9 1.3.5 C端multi-functional basic domain ------------------------------------------- 9 1.4 影響p53的上游訊息(Signals to p53) ------------------------------------------ 10 1.4.1 Translational regulation ------------------------------------------------------- 10 1.4.2 Post-translational modification ----------------------------------------------- 11 (1) Phosphorylation ----------------------------------------------------------------- 11 (2) Dephosphorylation ------------------------------------------------------------- 12 (3) Acetylation ----------------------------------------------------------------------- 12 1.4.3 Oncogenic regulation ---------------------------------------------------------- 13 1.4.4 Telomere shortening ----------------------------------------------------------- 13 1.5 p53所引發的下游訊息(Signaling out) ---------------------------------------- 14 1.5.1 p53對Cell Cycle的調控------------------------------------------------------- 14 1.5.2 p53對apoptosis的調控-------------------------------------------------------- 15 1.6 p53在維持基因體穩定所扮演的角色---------------------------------------- 18 1.7 p53 kingdom ------------------------------------------------------------------------ 19 1.7.1 The roles in DNA damaging signals ---------------------------------------- 19 1.7.2 The roles in development of embryo --------------------------------------- 20 1.7.3 Involvement in tumor suppression ------------------------------------------ 20 (貳)‧本論文研究的目的------------------------------------------------------------------ 22 (參)‧實驗材料與方法 3.1 材料------------------------------------------------------------------------------------ 23 3.1.1 化學藥品和實驗材料---------------------------------------------------------- 23 3.1.2 酵素和試劑----------------------------------------------------------------------- 23 3.1.3 質體DNA ------------------------------------------------------------------------- 24 3.1.4 cDNA ----------------------------------------------------------------------------- 24 3.1.5 細胞株---------------------------------------------------------------------------- 25 3.1.6 放射性物質---------------------------------------------------------------------- 25 3.1.7 選殖cDNA及genomic DNA的引子----------------------------------------- 25 (1) 選殖cDNA的引子--------------------------------------------------------------- 25 (2) 選殖genomic DNA的引子----------------------------------------------------- 26 3.2 方法------------------------------------------------------------------------------------ 28 3.2.1 細胞培養------------------------------------------------------------------------- 28 3.2.2 RNA的製備--------------------------------------------------------------------- 28 3.2.3 北方墨點轉漬分析(Northern blot analysis) ------------------------------ 30 3.2.4 質體製備------------------------------------------------------------------------- 32 3.2.5 DNA片段的選殖(cloning) -------------------------------------------------- 34 3.2.6 mRNA差異展現分析法(mRNA Differential Display) ----------------- 36 3.2.7 Rapid Amplification of cDNA 5''-Ends (5''RACE) ------------------------ 37 3.2.8 RNase protection assay ------------------------------------------------------- 37 3.2.9 PCR-based genome walking method --------------------------------------- 38 3.2.10 Primer extension method ---------------------------------------------------- 39 3.2.11 質體的構築--------------------------------------------------------------------- 40 3.2.12 細胞群落形成分析(Colony formation assay) --------------------------- 42 3.2.13 MTX uptake -------------------------------------------------------------------- 42 3.2.14 Luciferase assay --------------------------------------------------------------- 43 (肆)‧實驗結果與討論 4.1 p53下游基因之選殖 4.1.1 實驗結果------------------------------------------------------------------------- 45 4.1.2 討論------------------------------------------------------------------------------- 47 4.1.3 圖表------------------------------------------------------------------------------- 49 4.2 mDDA1基因之選殖與功能分析及受p53誘導之機制分析 4.2.1 實驗結果 (1) mDDA1 cDNA之選殖-------------------------------------------------------- 54 (2) mDDA1 genomic DNA之選殖---------------------------------------------- 56 (3) p53活化mDDA1表現之機制----------------------------------------------- 57 (4) mDDA1的生物功能分析---------------------------------------------------- 58 (5) mDDA1為thiamine transporter基因---------------------------------------- 59 4.2.2 討論 (1) p53誘導mTHTR-1表現之機制--------------------------------------------- 61 (2) mTHTR-1基因的生物功能角色------------------------------------------- 62 4.2.3 圖表------------------------------------------------------------------------------- 67 4.3 mDDA3基因之選殖與功能分析及受p53誘導之機制分析 4.3.1 實驗結果 (1) mDDA3 cDNA之選殖-------------------------------------------------------- 82 (2) mDDA3基因的genomic DNA之選殖------------------------------------- 83 (3) mDDA3 transcripts的5''-end heterogeneity成因之分析---------------- 83 (4) Heterogeneous mDDA3 cDNAs之分析----------------------------------- 84 (5) DDA3 transcript在老鼠和人類組織之表現分析----------------------- 85 (6) 以RNase protection和primer extension方法印證 mDDA3 mRNA的5''-end heterogeneity ------------------------------------ 85 (7) p53誘導mDDA3表現之機制----------------------------------------------- 87 (8) mDDA3的生物功能分析---------------------------------------------------- 88 (9) 人類DDA3 cDNA之選殖----------------------------------------------------- 89 4.3.2 討論------------------------------------------------------------------------------- 90 (1) Differential transcription initiation及alternative RNA splicing調控mDDA3表現之探討------------------------------------ 90 (2) mDDA3基因表現之調控---------------------------------------------------- 92 (3) mDDA3的生物功能角色---------------------------------------------------- 94 4.3.3 圖表------------------------------------------------------------------------------- 97 (伍)‧結論----------------------------------------------------------------------------------- 119 (陸)‧參考文獻----------------------------------------------------------------------------- 120 附錄 (一) 附圖------------------------------------------------------------------------------------ 139 (二) 英文論文 1. Identification of a novel mouse p53 target gene DDA3---------------------- 140 2. Transcriptional induction of a thiamine transporter gene by p53----------------------------------------------------------------------------------- 150

碩士<br>國防醫學院<br>生物及解剖學研究所<br>96<br>The p53 gene family consists of three genes, p53, p63, and p73, which encoding for sequence-specific nuclear transcription factors with high homology in their own DNA binding domain. These transcription factors could recognize the similar responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumor progression or developmental disease. It is well-known that genes have p53 RE in promoter region can be trans-activated by p53 family members. But the regulation mechanism of the p53 RE at enhancer region is still un-clarity. The p53 REs with different core sequence, different direction, and different length are important factors for trans-activation of the p53 family members. We proposed that the regulation of p53 RE sequence variants in enhancer region is different from the promoter region. Therefore, we established an in vitro model system for analysis transcriptional regulation by different p53 REs at promoter and enhancer regions. In addition, we also analyzed the different transcriptional responses regulated by different isotypes of p53 and p63 to find out the determinant factors of these REs. Furthermore, we used the online database to search the known and predicted RE sequence be regulated by p53 family to evaluate our model. Finally, we could use this in vitro model system to search the novel genes regulated by p53 family.

碩士<br>國立中央大學<br>生命科學研究所<br>88<br>p53 is a well-studied tumor suppressor gene. In the past, many conventional methods were used to identify p53 function-associated genes. In order to identified unknown genes whose function were related with p53, colormetric cDNA microarray were used to study the genome-wide transcriptional expression pattern of genes, which are regulated by tumor suppressor gene p53 in human non small cell lung cancer cell line H1299. To chase the downstream genes of p53, the cell line H1299-p53V173L was used for experiments since it expresses wild-type p53 once the growth temperature was shifted from 37℃ to 32℃. Post temperature shift from 37℃ to 32℃, cells were harvested at the following time intervals: 0, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours. The derived cDNA were labeled and hybridized to microarray membranes containing 9600 cDNA dots. The signal density of these cDNA dots were acquired with image processing for statistic analysis. Through such process, totally 144 genes were identified as p53-upregulated or p53-downregulated, and were further sequence verified. The majority of these genes are related with signal transduction, cell cycle, metabolic regulation and DNA repair. Some genes found associated with p53 in the literature were successfully identified, for instance, PCNA, ku80, APEX etc. According to the data in this thesis, p53 might control many genes expression, even though when cells were not stimulated by X-ray or hypoxia. Among those genes, the most interesting one is the MHC (major compatibility complex) class I, which plays a major role in immune response. Different alleles of MHC class I was observed significantly and consistently induced by p53. This indicates that p53 may be involved in some immune pathway to target stressed or tumor cells for elimination. The association between p53 and immune system was all the time totally ignored. With cDNA microarray technology, this association is confirmed and is worth with further investigation.