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Identification and characterization of TNFalpha responsive genes in human breast cancer cells
(2006)
One of the hallmarks of cancer is the escape of the transformed cells from apoptosis. Therefore, the identification of survival genes, allowing cancer cells to circumvent programmed cell death, could provide new diagnostic markers as well as targets for therapeutic intervention. A well known transcription factor regulating the balance between pro- and anti- apoptotic factors is NF-kappaB, which is strongly induced by tumor necrosis factor alpha (TNFalpha). When cells are stimulated by TNFalpha their response is biphasic with an initial NF-kappaB induction of survival genes which is overridden by the subsequent activation of initiator caspases triggering apoptosis. By combining gene trap mutagenesis with site specific recombination a strategy was developed, which enriches for genes induced by TNFalpha in the human breast cancer cell line MCF-7. The strategy relies on a one way gene expression switch based on Cre/loxP mediated recombination, which uncouples the expression of a marker gene from the trapped cellular promoter thereby enabling the recovery of genes that are only transiently induced by TNFalpha. The marker gene used in these experiments was a dominant negative variant of the TNFalpha-receptor associated protein FADD (dnFADD), which blocks the apoptotic branch of the TNFalpha induced signaling pathway. Initial experiments indicated that MCF-7 cells expressing high levels of dnFADD were insensitive to TNFalpha induced apoptosis and therefore suitable for the installment of a one way gene expression switch susceptible to Cre/loxP mediated recombination. A MCF-7 reporter clone harboring the recombinase dependent gene expression switch was infected with the gene trap retrovirus U3Cre, which inserts the Cre recombinase gene into a large collection of chromosomal sites. Insertion of Cre downstream of an active cellular promoter induces dnFADD expression from the gene expression switch enabling the cells to block TNFalpha triggered apoptosis. From a gene trap integration library containing approximately 2000000 unique proviral integrations, 69 unique TNFalpha inducible gene trap insertion sites were recovered in a two step selection procedure. Sequencing of the genomic regions adjacent to the insertion sites, which were obtained by inverse PCR (gene trap sequence tags, GTSTs), and data base analysis revealed that 42% of the GTSTs belonged to annotated genes, 13% to known cDNAs with open reading frames, 17% to Genscan predicted genes, 9% to ESTs, 9% to repetitive sequences and 10% to unannotated genomic sequence. Overall, 44% of the annotated genes recovered in this screen were directly or indirectly related to cancer, indicating that the gene trap strategy developed here is suitable for the identification of cancer relevant genes. Analysis of the expression patterns of the trapped and annotated genes in wild type cells revealed that 19 out of 24 genes were either up- or down- regulated by a factor of at least 1.45 by TNFalpha. A large fraction of the gene trap insertions were located upstream, in introns or in opposite orientation to annotated transcripts, indicating that the strategy efficiently recovers non-coding RNAs (ncRNAs). While the biological significance of these transcripts still needs to be elucidated, they fall into two main categories. The first category includes gene trap insertions upstream of genes, which could either represent regulatory RNAs interacting with promoter elements or transcripts driven by bidirectional promoters. The second includes inverse orientation gene trap insertions in introns of annotated genes suggesting the presence of natural antisense transcripts (NATs). Interestingly, more than 50% of all antisense integrations are located downstream of transcription start sites predicted by different algorithms supporting the existence of RNAs transcribed from the corresponding genomic regions. Intronic integrations on the coding strand could be derived from cryptic splicing, alternative promoter usage or additional, so far uncharacterized transcripts. Preliminary functional analysis of two genes recovered in this screen encoding the transcription factor ZFP67 and the FLJ14451 protein revealed that FLJ14451 but not ZFP67 inhibited anchorage independent growth in soft agar, suggesting that FLJ14451 might have some tumor suppressor functions. In summary, besides identifying a putative tumor suppressor protein, the present experiments have shown that gene trapping is useful in identifying non-coding transcripts in living cells and may turn out to be the method of choice in characterizing these transcripts whose functions are still largely unknown.
Alzheimer’s Disease (AD) is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterised by the presence of amyloid plaques, intracellular neurofibrillary tangles and pronounced cell death. The aim of this thesis was to investigate pathways involved in the Aß cascade of neurodegeneration. Since novel findings indicate that already this Aß species exerts neurotoxic effects long before hyperphosphorylated tau, neurofibrillary tangles and extracellular Aß plaques appear, the investigations were accomplished with specific regard to the effects of intracellular Aß. The Swedish double mutation in the APP gene results in six- to eightfold increased Aß production of both Aß1-40 and Aß1-42 compared to human wildtype APP cells (APPwt). Data obtained from PC12 cells indicate that it is possible to specifically increase the Aß load without enhancing APP expression levels. On the basis of these findings, it seemed possible to investigate dose-dependent effects of Aß in multiple experimental designs. These assay designs were created in order to mimick different in-vivo situations that are discussed to occur in AD patients: APPsw PC12 cells exhibit low physiological concentrations of Aß within picomolar range in contrast to APPsw HEK cells, expressing Aß levels within the nanomolar range. Of note, the APPsw HEK cells showed a specific and highly significant increase in the intracellular accumulation of insoluble Aß1-42. Moreover, an intracellular accumulation of Aß and APP was found in the mitochondria of the HEK APPsw cells suggesting a direct impact on mitochondrial function on these cells. This effect might finally lead to disturbances in the energy metabolism of the cell or to increased cell death. Furthermore, baseline g- and ß-secretase activity was assessed since these enzymes represent promising therapeutic targets to slow or halt the disease process. As expected, ß-secretase activity was significantly elevated in all APPsw cell lines. This might be due to the proximity of the Swedish double mutation next to the N-terminus of the Aß sequence. Interestingly, g-secretase activity was similarly increased in PC12 APPsw cells. In addition, the toxicity of different Aß species was investigated in SY5Y and PC12 cells with regard to their effect on cellular viability mirrored by mitochondrial activity using MTT assay. Here, it turned out that not monomers, but already dimers are neurotoxic correlates. Fibrillar Aß species showed the highest toxicity. In the next step, SY5Y cells forming endogenous, dimeric APP and Aß were investigated. In accordance with previous findings, these cells showed a decreased MTT reduction potential in comparison to APPwt and control SY5Y cells reflecting a decrease of cellular viability. The impaired energy metabolism of the cells was even more drastically mirrored by reduced baseline ATP levels. In the second part of this thesis, the expression and intracellular distribution of Bcl-2 family proteins and pro-apoptotic mitochondrial factors under baseline conditions and during oxidative stress were analyzed in the APPwt and APPsw bearing cells. The most prominent finding was the reduction of expression levels of the anti-apoptotic factor Bcl-xL in the cytosolic fractions of APPwt and APPsw PC12 cells. This might indicate that a lack of anti-apoptotic factors or their altered intracellular distribution, rather than an increase in caspase-dependent pro-apoptotic factors, could be responsible for the increased vulnerability of APPwt- and APPsw-transfected PC12 cells against oxidative stress. Since total Bcl-xL expression was unaffected in PC12 cells, in contrast to APPwt and APPsw-expressing SY5Y and HEK cells revealing significantly decreased Bcl-xL expression levels. Thus, alterations in Bcl-xL distribution seem to be an early event in the disease process. Increasing Bcl-xL expression might potentially be one promising strategy for AD modification. PC12 and HEK cells bearing APPsw or APPwt were treated with the potent g-secretase inhibitor DAPT. Of note, DAPT did not only efficiently block Aß production, but additionally led to an elevation of the MTT reduction potential, reflecting an increase in cellular viability. As another disease-modifying strategy, several efforts are undertaken to ameliorate AD-relevant symptoms by the treatment with nerve growth factor (NGF). Generally, it is known that substituted pyrimidines have modest growth-promoting effects. Here, KP544, a novel substituted pyrimidine, was characterised. This drug increased MTT reduction potential in terminally differentiated and undifferentiated PC12 cells. Furthermore, treatment with KP544 led to a reduction in Aß1-40 secretion. Thus, one may conclude that the target of KP544, GSK-3ß, represents a connecting link between the two main pathological hallmarks of AD and might thus be a very promising therapeutic target for AD.