Multidrug resistance (MDR) in small cell lung cancer is one of the major causes of failures of chemotherapy. MDR is a means of protection of tumor cells against chemotherapeutic drugs. Although the molecular basis of MDR is not fully understood, genes involved in apoptosis may be mutated. Recent finding of a link between over-expression of an apoptotic gene, cyclooxygenase 1 (cox 1), and MDR suggests that cox 1 is involved in the development of MDR phenotype. This research was an attempt to observe whether up-regulation of cox 1 contributes to the MDR phenotype in small cell lung cancer cells. This research ultimately may provide a mechanism to reverse the abberant up-regulation of apoptosis genes associated with multidrug resistance to either eliminate or control reproduction of cancer cells. Real time RT PCR was used to confirm the up-regulation of cox 1 in cultured MDR resistant small cell lung cancer cells (GLC4). The up-regulated cox 1 expression was down-regulated using RNA interference technology (RNAi) by transfection with an anti-cox 1 siRNA. More than 90% transfection of cells was confirmed using confocal microscopy. Down-regulation of cox 1 was validated as the protein expression significantly decreased (P=0.004) from multidrug resistant small cell lung cancer transfected cells compared to multidrug resistant nontransfected cells. There was decrease level of expression of cox 1 in multidrug resistant cells after the knockdown with siRNA specific to cox 1. The decreased level of cox 1 expression and, therefore, Cox 1 production increased the rate of apoptosis in small cell lung cancer cells as indicated by its sensitivity to the doxorubicin. / Department of Biology

The gene, cox-1, is over-expressed in cultured GLC4 small cell lung cancer cells concurrent with the development of multi-drug resistance (MDR) as a result of the use of the chemotherapeutic agent used to combat the cancer, doxorubicin. Prevention of MDR has been a tremendous challenge in cancer research and this research is concerned with abolishment of MDR as a cancer survival strategy. RNA-mediated interference technology (RNAi) was employed using siRNA to decrease cox-1 expression and temporarily restore the susceptibility of the cells to doxorubicin. GLC4 cells are of three types: S (sensitive cells never exposed to doxorubicin); ADR (MDR cells cultured in doxorubicin), and; REV (revertant cells previously cultured in presence of doxorubicin but no longer). REV and ADR cells were transfected with cox-1 siRNA. After 24 h, 1x106cells were used for RNA isolation and 1 μg of RNA was used for RT-PCR to assess down-regulation of cox-1 RNA. RT-PCR results indicated that cox-1 RNA was down-regulated to basal levels seen before exposure to doxorubicin. Ct values for GLC4/ADR and cox-1 down-regulated GLC4/ADR cells were 23 and 34, respectively. The result indicated abundant levels and moderate levels of cox-1 mRNA in the ADR cells and the transfected ADR cells respectively. The relative expression level of cox-1 mRNA was 33% higher in the non-transfected GLCR/ADR cells as compared to the transfected GLCR/ADR cells as shown by the curve. Two hundred thousand cells were used for hemacytometer cell counts in the presence of trypan blue to assess cell viability. cox-1 down-regulation in ADR cells resulted in a significantly higher percentage of non-viable cells (25.4%) as compared to its non-transfected control (20.5%) using a Student’s t-test (*P <0.05). Similarly, fluorescence microscopy confirmed that apoptosis was significantly increased in the ADR cells treated with doxorubicin and cox-1 siRNA simultaneously (69.4%) as compared to its non-transfected control (56.7%) (*= P <0.01). A Western blot analysis performed by Fernando Cuadrado indicated that siRNA transfection decreased the expression of COX-1 by 66% in GLC4/ ADR cells as compared to the non-transfected control using densitometry. However, no conclusive results were obtained using flow cytometry as the flow cytometer was incapable of analyzing the mixed cell population (adherent and suspension) which is a characteristic of this cell line, GLC4. Thus, we have clearly demonstrated that MDR cancer cells can be altered temporarily to become susceptible to doxorubicin, a potentially important finding for the treatment of cancer patients. / Department of Biology

Romantic relationships are one of the most important relationships a woman will develop in her life. Women are often socialized to be compliant within their relationships, and are reminded of how a thin body type is ideal. The implications of this socialization can affect the way a woman feels about her body. The current study included 207 women who ranged in age from 18-30, were in a relationship (neither married nor engaged) for 6 months or longer, and identified as either being attracted to the same or opposite sex. Results of the study indicated a significant effect of higher levels of body dissatisfaction between both negative relationship quality, and increased engagement in unhealthy dietary behaviours. In addition, self-silencing was found to be a significant mediator in the relationship between relationship quality and both body dissatisfaction and unhealthy dietary behaviours. Limitations of the study and directions for future research are discussed.

Romantic relationships are one of the most important relationships a woman will develop in her life. Women are often socialized to be compliant within their relationships, and are reminded of how a thin body type is ideal. The implications of this socialization can affect the way a woman feels about her body. The current study included 207 women who ranged in age from 18-30, were in a relationship (neither married nor engaged) for 6 months or longer, and identified as either being attracted to the same or opposite sex. Results of the study indicated a significant effect of higher levels of body dissatisfaction between both negative relationship quality, and increased engagement in unhealthy dietary behaviours. In addition, self-silencing was found to be a significant mediator in the relationship between relationship quality and both body dissatisfaction and unhealthy dietary behaviours. Limitations of the study and directions for future research are discussed.

A major contributor to mortality in patients with plasma cell dyscrasias (PCDs); i.e., multiple myeloma, light chain deposition disease and AL amyloidosis is the deposition as insoluble aggregates of monoclonal immunoglobulin light chain proteins (LC) in the kidneys and other organs. Currently anti-plasma cell chemotherapies are used to reduce LC synthesis, and slow deposition. While effective, these treatments are toxic, non-specific, expensive, and might not be appropriate in all cases, making the identification of an alternate means of reducing toxic LC species desirable. To this end, we have investigated whether RNA interference (RNAi) could achieve these goals. Human (RPMI 8226, Bur) and transfected mouse myeloma (SP2/O-lambda 6) cells which produce measureable quantities of human LC protein were used as model systems for testing the efficacy of both synthetic small interfering RNAs (siRNAs) and short hairpin RNA (shRNA) expression vectors in reducing LC synthesis. Sequencing of LC genes provided the basis for design of siRNA duplexes targeting either the variable (V) or joining (J) regions of individual LCs, or the constant (C) region of either kappa or lambda LC isotypes. Myeloma lines were transfected with siRNAs using lipid-based transfection media. Cells receiving non-silencing siRNAs served as controls. Exposure of myeloma lines to siRNAs was well tolerated and no cytotoxicity was observed. LC mRNA expression was shown to be reduced ≥40% in 8226 and SP2/O- lambda 6 cell lines receiving siRNA treatment as compared with untreated controls. Exposure to siRNAs was also effective in significantly reducing both intracellular and secreted LC protein levels in cell lines tested as evidenced by flow cytometry or enzyme-linked immunosorbent assays (ELISAs). Effective siRNA nucleotide sequences were used to generate shRNA cassettes which were ligated into lentiviral expression vectors under the control of the RNA polymerase III promoter, U6. These expression systems were used to generate replication incompetent lentiviral particles. Exposure of 8226 to lentiviral particles resulted in significant knockdown of LC mRNA and protein both in vitro and in xenograft tumor bearing immune compromised mice. These results provide positive evidence for the ability of RNAi based approaches to reduce LC secretion in models of PCD.

It is well established that epigenetic modifications to the genome are crucial for the exquisite control of gene expression required for an organism to develop and differentiate. These modifications are maintained through mitotic rounds of cell division, but must be cleared and reset through meiosis in order for the cells of the early embryo to achieve totipotency. Although we know these mechanisms exist, the rules determining which modifications are established where on the genome and the genes involved in these processes remain poorly characterised. Much of what is known about epigenetic processes has come from studies in non-mammalian organisms, such as Drosophila. However, in our laboratory we have developed a mammalian system for identifying modifiers of epigenetic gene silencing. An ENU mutagenesis screen is being carried out using an inbred mouse line carrying a GFP transgene, with an erythroid-specific promoter, that is particularly sensitive to changes in epigenetic modifications. Currently, 14 mutant lines that display a heritable shift in GFP expression have been recovered. These have been termed Modifiers of Murine Metastable Epialleles (Mommes). When I began my PhD in 2005, we had not identified any of the mutations underlying the phenotypes observed. To confirm the efficacy of the screen, I have tested the effect of heterozygosity for null alleles of two known epigenetic modifiers, Dnmt3a and Dnmt3b, on expression of the GFP transgene. Heterozygosity for the Dnmt3b knockout allele does shift expression while heterozygosity for the Dnmt3a knockout allele does not. This highlights the limitations of the screen. With this particular screen we will only detect modifiers that are expressed during haematopoiesis in the bone marrow. I have also worked on MommeD5. MommeD5 is a semi-dominant, homozygous embryonic lethal mutation that acts as an enhancer of variegation. I have found that the MommeD5 allele carries a 7 bp deletion in the major histone deacetylase, Histone deacetylase 1 (Hdac1), and this significantly alters the C-terminus of the mutant protein. The finding of Hdac1 attests to the screen design. The MommeD5 homozygous mutants die at approximately the same time as the published knockout of Hdac1 and the heterozygous mutants show increased levels of Hdac2 and acetylated histone H3, as reported in Hdac1-deficient embryonic stem cells. In addition, I have studied the effect of heterozygosity for each of the mutations on the phenotype of the mouse. In general, heterozygous Momme mutants are viable and fertile, but show subtle abnormal phenotypes. However, in the case of MommeD5 none were observed and this may relate to the compensatory upregulation of other histone deacetylases. In the case of Dnmt3a and Dnmt3b a sex ratio distortion is seen in the colonies, with less males seen than expected. Also, Dnmt3a heterozygous mutant males that inherited the mutant allele from the dam are smaller and show an increased range of body weights compared to their wild-type male littermates. This may be an example of intangible variation, i.e. phenotypic variation observed in isogenic individuals raised in standardised environments. These results suggest that epigenetic mechanisms have a role in intangible variation, also known as developmental noise. Despite the fact that it is now acknowledged by many that stochastic events occur at the level of the cell, the idea that it can happen at the level of the whole organism is rarely considered.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 137-151). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.