Dissertations / Theses on the topic 'P53-regulated genes'

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.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Faculty of Science, Environment, Engineering and Technology
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
國立陽明大學
生物化學研究所
93
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
國立陽明大學
生物化學研究所
88
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
國防醫學院
生物及解剖學研究所
96
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
國立中央大學
生命科學研究所
88
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.

碩士
國立陽明大學
生物化學研究所
87
Wild type p53 expressed from a temperature-sensitive (tsp53) construct induces both G1 cell cycle arrest and apoptosis in the p53-negative IW32 mouse erythroleukemia cell line. Using PCR-based differential display analysis, we previously identified a new p53-inducible gene, DDA1, whose mRNA was upregulated in tsp53-transfected IW32 cells following induction of wild type p53 expression by temperature shift to 32°C. The DDA1 mRNA induction was detectable within 1 hour after temperature downshift, and rapid degradation was observed when the temperature was shifted back to 37°C, suggesting that the expression of DDA1 is dependent on the continuous presence of p53. The DDA1 mRNA was also induced in DNA damaging reagent-treated NIH3T3 cells. Previous studies from this lab have shown that mouse DDA1 cDNA predicted to encode a protein of 498 amino acid residues containing 12 transmembrane domains. The loop region between 6th and 7th transmembrane domains was used as immunogen to produce rabbit polyclonal antibodies, and antibodies that can recognize E. coli expressed mDDA1 protein was obtained. Immunofluorescence analysis indicated that DDA1 protein is located in the cytoplasm. Overexpression of mDDA1 cDNA in H1299 cells inhibited cell growth，as shown by the colony formation assay. The cDNA of human DDA1 that is 73 % identical to mouse DDA1 was acquired by library screening and database searching. The two amino acid sequences share 90 % identity.

碩士
國立陽明大學
生物化學研究所
88
Abstract p53 tumor suppressor is a transcription factor that causes cell growth arrest and induces apoptosis. Identification of the p53 downstream target genes is therefore important to unravel the mechanisms underlying p53 actions. We have previously identified and cloned a p53-regulated gene, DDA1, by the RNA differential display of an IW32 erythroleukemia stable clone (1-5) that contains a temperature-sensitive p53 mutant gene, tsp53val135. 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). To further investigate the subcellular localization and function of DDA1, we established DDA1 expressing clones under the control of the tetracycline inducible promoter. After 24 hours treatment of 2 mg /ml Doxycycline, clone 118 transfectant cells could be induced to express DDA1 mRNA and protein. Immunofluorescence analysis indicated that DDA1 was present on the plasma membrane. Growth of DDA1 stable transfectant was partly inhibited in the presence of Doxycycline. Ability of the clone 118 cells to uptake thiamine increased 2-fold in the presence of Doxycycline. These data demonstrated that mDDA1 possesses thiamine transporter activity. hTHTR-1 mRNA was induced by DNA damage in a p53 dependent manner. Induction was detected in 293 cells expressing endogenous p53, but not in 293T cells whose p53 was inactivated. Together these results indicate that the high affinity thiamine transpoter is a p53 regulated gene. 中文摘要 ..................................................... 1 英文摘要 ..................................................... 2 緒論 ..................................................... 3 實驗材料 ..................................................... 16 實驗方法 ..................................................... 18 實驗結果 ..................................................... 30 實驗討論 ..................................................... 36 參考文獻 ..................................................... 40 附圖 ..................................................... 47

碩士
國立陽明大學
生物化學研究所
86
p53為一抑癌基因，當細胞DNA損害時p53常會大量表現導致G1 arrest或apoptosis。由本實驗室所建立的可持續表現受溫度調控的p53 mutant的轉型細胞株1-5，利用而tNA差異展現法(differential display)我們選殖到可受p53調控的基因mDDA3。經DNA序列分析，發現血DDA3的cDNA全長為1.8kb。再由human EST database找到了一個EST clone將其定序並與老鼠的DDA3比對後，發現二者之胺基酸序列具有67.9%的相似性，並且皆含3個SH3 binding motif PXXP。Colony的rmation assay結果顯示DDA3大量表現可以抑制H1299人類肺癌細胞的生長。將mDDA3的N端接上FLAGpeptides，以immunofluorescence觀察可見而JDA3在細胞質中表現。在invitro及in vivo下做轉錄和轉譯，可得大小約39kD和43kD的蛋白，這和由DDA3的open reading frame所預測出的蛋白質大小接近。利用human及mouse DDA3的相似區域我們設計了二條peptides，將其與BSA結合後注射兔子可以測得血清中有抗體。至於其與DDA3蛋白之結合，則有待更進一步的實驗確認。 p53 tumor suppressor is a transcription factor that functions through activation of a number of downstream target genes. We have previously identified and cloned a p53 regulated gene, DDA3, through differential display of the mRNAs of the IW32 mouse erythroleukemia cells grown in the presence or absence of wild type p53. The mouse DDA3 cDNA (mDDA3) is about 1.8 kb in size. Two alternatively spliced transcripts were cloned, mDDA3s contains an open reading frame of 329 amino acids and mDDA3L has an additional ATG at 45 bp upstream of the translation initiation codon of mDDA3s. Through homology search of the human EST database, an EST clone was identified and sequenced. This cDNA showed 69% identity in protein sequence with mDDA3, and may represent the human homologue of mDDA3. In vitro transcription and translation showed that mDDA3 and hDDA3 could both encode proteins of 39kDa in size. When mDDA3 was fused to the Flag tag at its 5' terminus and introduced into cells, a protein of 43kDa that reacted with anti-Flag antibody was found. Immunofillorescence indicates that DDA3 was distributed in the cytosol. Over-expression of DDA3 suppressed the growth of H1299 human non-sinall-cell lung cancer cells, as analyzed by colony formation assay. These results suggest that DDA3 may mediate, at least in part, the growth suppressing function of p53.

碩士
國立陽明大學
生物化學研究所
87
Abstract p53 tumor supressor is a transcription factor that causes cell growth arrest and induces apoptosis, identification of the p53 downstream target genes is therefore important to unravel the mechanisms underlying p53 actions. We have previously identified and cloned a p53-regulated gene, DDA3, by the RNA differential display of an IW32 erythroleukemia stable clone (1-5) that contains a temperature-sensitive p53 muntant gene, tsp53 val135. The induction of DDA3 mRNA could be detected within 2 hours and DDA3 mRNA rapidly degraded when the temperature was shifted back to 38℃, suggesting that the expression of DDA3 is dependent on wild type p53. Induction of DDA3 mRNA was also observed in DNA damaging reagent-treated NIH3T3 cells. Previous studies revealed that mouse DDA3 genome contains consensus AP-1 and cAMP responsive elements, however, no induction of DDA3 mRNA was detected when NIH3T3 cells were treated with TPA or 8-Br cAMP. In order to understand the relationship between the DDA3 expression and cell cycle progression, clone 1-5 cells were exposed for 16h to mimosine (G1 phase arrest), thymidine (S phase arrest) and nocodazole (G2/M phase arrest) before shifting to 32℃. DDA3 mRNA induction was found only in cells treated with thymidine, suggesting cell cycle-dependent expression of DDA3. Immunofluorescence analysis showed that DDA3 protein was expressed in the cytoplasm. To explore the biological functions of DDA3, we have established several tetracyclin inducible DDA3 expression cell clones. Maximal induction of DDA3 mRNA could be reached after doxycyclin treatment. Analysis by flow cytometry and serum starvation indicated that DDA3 expression did not change the cell cycle distribution or growth factor dependency of these cells. The biological significance of DDA3 induction by p53 remains to be elucidated.

博士
國立陽明大學
生物化學研究所
90
The p53 tumor suppressor is a transcription factor that activates the expression of many target genes. We have previously reported the identification of a p53-regulated mouse gene DDA3 (mDDA3). The 5’ upstream genomic region of the mouse DDA3 was cloned, and sequence analysis revealed the presence of a potential p53 response element (RE2) residing at nucleotides +390 ~ +409 relative to the first translation start site. When fused upstream to a luciferase reporter gene, 5’ genomic regions of the mDDA3 gene containing RE2 were shown to be responsive to the wild-type, but not mutant p53, in a transient transfection assay. RE2 was sufficient to confer the transactivation responsiveness to p53. Furthermore, gel mobility shift analysis showed that RE2 formed specific complexes with wild-type p53. Induction of mDDA3 was found in adriamycin treated normal mouse embryonic fibroblast cells (MEF), but not in p53 knockout (p53-/-) MEF. Overexpression of p73 induced mDDA3 mRNA expression, and luciferase reporter analysis indicated that RE2 was responsive to transactivation by members of the p73 family proteins. Consistent with these findings, elevated expression of p73 protein and mDDA3 mRNA were observed concomitantly in the p53-/- MEF cells treated with cisplatin. These results together demonstrated that mDDA3 is a transcriptional target gene of p53 and its related-protein p73. The mDDA3 protein was localized to the cytoplasm, while upon adriamycin-induced DNA damage, the protein was translocated to the nucleus. Western blot analysis on mouse tissues revealed specific expression of mDDA3 in the brain. Immunocytochemistry studies demonstrated the distribution of mDDA3 in various regions of the brain, with prominent expression localized to the hypothalamus. Similar patterns of mDDA3 expression were found in the p53 knockout mice, suggesting that its expression in the brain is governed by a p53-independent mechanism. Using two independent cell lines, the tsp53val135 transfected IW32 mouse erythroleukemia cells and NIH3T3 cells, we showed that mDDA3 was induced in the S and G2 phases of the cell cycle. The cell cycle-dependent expression was mediated through the RE2 sequence, suggesting the involvement of p53 or its family members. Cotransfection of mDDA3 with LacZ suppressed the number of cells expressing -galactosidase, suggesting that mDDA3 expression inhibits cell growth. Taken together, this study has clearly demonstrated that mDDA3 is a direct transcriptional target gene of p53 and p73. Furthermore, the expression of mDDA3 in cells is regulated by cell cycle machinery in a p53-dependent manner. The functional significance of mDDA3 expression in the brain remains to be elucidated.

publisher version
The Hsp70/Hsp90 organising protein (HOP) is a co-chaperone essential for client protein transfer from Hsp70 to Hsp90 within the Hsp90 chaperone machine. Although HOP is upregulated in various cancers, there is limited information from in vitro studies on how HOP expression is regulated in cancer. The main objective of this study was to identify the HOP promoter and investigate its activity in cancerous cells. Bioinformatic analysis of the -2500 to +16 bp region of the HOP gene identified a large CpG island and a range of putative cis-elements. Many of the cis-elements were potentially bound by transcription factors which are activated by oncogenic pathways. Luciferase reporter assays demonstrated that the upstream region of the HOP gene contains an active promoter in vitro. Truncation of this region suggested that the core HOP promoter region was -855 to +16 bp. HOP promoter activity was highest in Hs578T, HEK293T and SV40- transformed MEF1 cell lines which expressed mutant or inactive p53. In a mutant p53 background, expression of wild-type p53 led to a reduction in promoter activity, while inhibition of wild-type p53 in HeLa cells increased HOP promoter activity. Additionally, in Hs578T and HEK293T cell lines containing inactive p53, expression of HRAS increased HOP promoter activity. However, HRAS activation of the HOP promoter was inhibited by p53 overexpression. These findings suggest for the first time that HOP expression in cancer may be regulated by both RAS activation and p53 inhibition. Taken together, these data suggest that HOP may be part of the cancer gene signature induced by a combination of mutant p53 and mutated RAS that is associated with cellular transformation.

碩士
國立交通大學
生物科技系所
102
It is highly desired to develop a rapid and sensitive detection system for analyzing protein-DNA interaction. For developing such detection system, it is important to select appropriate protein carrier and good indicator. Nanodiamond (ND) is one of the biocompatible nanomaterials with large tunable surface for chemical modification. It possesses unique mechanical, spectroscopy, and thermal conducting properties. It is an excellent molecular vehicle to deliver specific molecules in biological system. The green fluorescent protein (GFP) is a protein that emits strong green fluorescence when it is excited by ultra-violet to blue light. It makes GFP a good indicator. By combining ND-GFP, a visible biocompatible delivery system will be developed. p53 is a tumor suppressor protein encoded by the TP53 gene. p53 plays an important role in apoptosis, genomic stability, and inhibition of angiogenesis by interacting with specific DNA sequence of promoter of related genes. In this study, a p53 functionalized ND-GFP DNA detecting probe will be developed. This complex can recognize the specific DNA sequence of promoter and the intermolecular interactions can be monitored directly by fluorescence and Raman spectroscopy both in vivo and in vitro.