Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection.

The beta 1-6 structure of N-linked oligosaccharides, formed by beta-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding beta-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of beta 1-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III. Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in beta 1-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for beta 1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.

Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation.

Transcription factor NF-E2 activity is thought to be crucial for the transcriptional regulation of many erythroid-specific genes. The three small Maf family proteins (MafF, MafG, and MafK) that are closely related to the c-Maf protooncoprotein constitute half of the NF-E2 activity by forming heterodimers with the large tissue-restricted subunit of NF-E2 called p45. We have established and characterized murine erythroleukemia cells that conditionally overexpress MafK from a metallothionein promoter. The conditional expression of MafK caused accumulation of hemoglobin, an indication of terminal differentiation along the erythroid pathway. Concomitantly, DNA binding activities containing MafK were induced within the MafK-overexpressing cells. These results demonstrate that MafK can promote the erythroid differentiation program in erythroleukemia cells and suggest that the small Maf family proteins are key regulatory molecules for erythroid differentiation.

Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain.

The c-myb protooncogene encodes a highly conserved transcription factor that functions as both an activator and a repressor of transcription. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus encode proteins that are truncated at both the amino and the carboxyl terminus, deleting portions of the c-Myb DNA-binding and negative regulatory domains. This has led to speculation that the deleted regions contain important regulatory sequences. We previously reported that the 42-kDa mitogen-activated protein kinase (p42mapk) phosphorylates chicken and murine c-Myb at multiple sites in the negative regulatory domain in vitro, suggesting that phosphorylation might provide a mechanism to regulate c-Myb function. We now report that three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb comigrate with three tryptic phosphopeptides derived from metabolically labeled c-Myb immunoprecipitated from murine erythroleukemia cells. At least two of these peptides are phosphorylated on serine-528. Replacement of serine-528 with alanine results in a 2- to 7-fold increase in the ability of c-Myb to transactivate a Myb-responsive promoter/reporter gene construct. These findings suggest that phosphorylation serves to regulate c-Myb activity and that loss of this phosphorylation site from the v-Myb proteins may contribute to their transforming potential.

Tumorigenic activity of the BCR-ABL oncogenes is mediated by BCL2.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.

Study of interaction between Si(O,C) nanowires and the biological system

Nanomedicine is a new branch of medicine, based on the potentiality and intrinsic properties of nanomaterials. Indeed, the nanomaterials ( i.e. the materials with nano and under micron size) can be suitable to different applications in biomedicine. The nanostructures can be used by taking advantage of their properties (for example superparamagnetic nanoparticles) or functionalized to deliver the drug in a specific target, thanks the ability to cross biological barriers. The size and the shape of 1D-nanostructures (nanotubes and nanowires) have an important role on the cell fate: their morphology plays a key role on the interaction between nanostructure and the biological system. For this reason the 1D nanostructure are interesting for their ability to mime the biological system. An implantable material or device must therefore integrate with the surrounding extracellular matrix (ECM), a complex network of proteins with structural and signaling properties. Innovative techniques allow the generation of complex surface patterns that can resemble the structure of the ECM, such as 1D nanostructures. NWs based on cubic silicon carbide (3C-SiC), either bare (3C-SiC NWs) or surrounded by an amorphous shell (3C-SiC/SiO2 core/shell NWs), and silicon oxycarbide nanowires (SiOxCy NWs) can meet the chemical, mechanical and electrical requirements for tissue engineering and have a strong potential to pave the way for the development of a novel generation of implantable nano-devices. Silicon oxycarbide shows promising physical and chemical properties as elastic modulus, bending strength and hardness, chemical durability superior to conventional silicate glasses in aggressive environments and high temperature stability up to 1300 °C. Moreover, it can easily be engineered through functionalization and decoration with macro-molecules and nanoparticles. Silicon carbide has been extensively studied for applications in harsh conditions, as chemical environment, high electric field and high and low temperature, owing to its high hardness, high thermal conductivity, chemical inertness and high electron mobility. Also, its cubic polytype (3C) is highly biocompatible and hemocompatible, and some prototypes of biomedical applications and biomedical devices have been already realized starting from 3C-SiC thin films. Cubic SiC-based NWs can be used as a biomimetic biomaterial, providing a robust and novel biocompatible biological interface . We cultured in vitro A549 human lung adenocarcinoma epithelial cells and L929 murine fibroblast cells over core/shell SiC/SiO2, SiOxCy and bare 3C-SiC nanowire platforms, and analysed the cytotoxicity, by indirect and direct contact tests, the cell adhesion, and the cell proliferation. These studies showed that all the nanowires are biocompatible according to ISO 10993 standards. We evaluated the blood compatibility through the interaction of the nanowires with platelet rich plasma. The adhesion and activation of platelets on the nanowire bundles, assessed via SEM imaging and soluble P-selectin quantification, indicated that a higher platelet activation is induced by the core/shell structures compared to the bare ones. Further, platelet activation is higher with 3C-SiC/SiO2 NWs and SiOxCyNWs, which therefore appear suitable in view of possible tissue regeneration. On the contrary, bare 3C-SiC NWs show a lower platelet activation and are therefore promising in view of implantable bioelectronics devices, as cardiovascular implantable devices. The NWs properties are suitable to allow the design of a novel subretinal Micro Device (MD). This devices is based on Si NWs and PEDOT:PSS, though the well know principle of the hybrid ordered bulk heterojunction (OBHJ). The aim is to develop a device based on a well-established photovoltaic technology and to adapt this know-how to the prosthetic field. The hybrid OBHJ allows to form a radial p–n junction on a nanowire/organic structure. In addition, the nanowires increase the light absorption by means of light scattering effects: a nanowires based p-n junction increases the light absorption up to the 80%, as previously demonstrated, overcoming the Shockley-Queisser limit of 30 % of a bulk p-n junction. Another interesting employment of these NWs is to design of a SiC based epicardial-interacting patch based on teflon that include SiC nanowires. . Such contact patch can bridge the electric conduction across the cardiac infarct as nanowires can ‘sense’ the direction of the wavefront propagation on the survival cardiac tissue and transmit it to the downstream surivived regions without discontinuity. The SiC NWs are tested in terms of toxicology, biocompatibility and conductance among cardiomyocytes and myofibroblasts.

Avaliação na linhagem endotelial tEnd dos efeitos diretos da transferência gênica de IFNbeta e p19arf e efeitos parácrinos mediados pela linhagem B16 transduzida pelos mesmos vetores adenovirais

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais

L’importance de la coopération de TRAF1 et LSP1 en aval du récepteur 4-1BB(CD137) pour la survie des lymphocytes

4-1BB (CD137) est un membre de la superfamille TNFR qui est impliqué dans la transmission des signaux de survie aux lymphocytes. TRAF1 est une protéine adaptatrice qui est recrutée par 4-1BB et autres TNFRs et est caractérisée par une expression très restreinte aux lymphocytes, cellules dendritiques et certaines cellules épithéliales. TRAF1 est nécessaire pour l’expansion et la survie des cellules T mémoire en présence d'agonistes anti-4-1BB in vivo. De plus, TRAF1 est requise en aval de 4-1BB pour activer (phosphoryler) la MAP kinase Erk impliquée dans la régulation de la molécule pro-apoptotique Bim. Suite à l’activation du récepteur 4-1BB, TRAF1 et ERK sont impliqués dans la phosphorylation de Bim et la modulation de son expression. L’activation et la régulation de TRAF1 et Bim ont un rôle important dans la survie des cellules T CD8 mémoires. Dans cette étude, nous avons utilisé une approche protéomique afin de pouvoir identifier de nouveaux partenaires de liaison de TRAF1. Utilisant cette stratégie, nous avons identifié que LSP1 (Leukocyte Specific Protein 1) est recruté dans le complexe de signalisation 4-1BB de manière TRAF1 dépendante. Une caractérisation plus poussée de l’interaction entre TRAF1 et LSP1 a montré que LSP1 lie la région unique N-terminal de TRAF1 de façon indépendante de la région conservée C-terminal. À l’instar des cellules T déficientes en TRAF1, les cellules T déficientes en LSP1 ne sont pas capables d’activer ERK en aval de 4-1BB et par conséquent ne peuvent pas réguler Bim. Ainsi, TRAF1 et LSP1 coopèrent en aval de 4-1BB dans le but d’activer ERK et réguler en aval les niveaux de Bim dans les cellules T CD8. Selon la littérature, le récepteur 4-1BB n’est pas exprimé à la surface des cellules B murines, mais le récepteur 4-1BB favorise la prolifération et la survie des cellules B humaines. Cependant, il est important d'étudier l'expression du récepteur 4-1BB dans les cellules B murines afin de disposer d'un modèle murin et de prédire la réponse clinique à la manipulation de 4-1BB. En utilisant différentes stimulations de cellules B murines primaires, nous avons identifié que le récepteur 4-1BB est exprimé à la surface des cellules B de souris suite à une stimulation avec le LPS (Lipopolysaccharides). Une caractérisation plus poussée a montré que le récepteur 4-1BB est induit dans les cellules B murines d'une manière dépendante de TLR4 (Toll Like Receptor 4). Collectivement, notre travail a démontré que la stimulation avec le LPS induit l’expression du récepteur 4-1BB à la surface des cellules B murines, menant ainsi à l'induction de TRAF1. De plus, TRAF1 et LSP1 coopèrent en aval de 4-1BB pour activer la signalisation de la Map kinase ERK dans les cellules B murines de manière similaire aux cellules T. Les cellules B déficientes en TRAF1 et les cellules B déficientes en LSP1 ne sont pas en mesure d'activer la voie ERK en aval de 4-1BB et montrent un niveau d’expression du récepteur significativement diminué comparé aux cellules B d’une souris WT. Ainsi, TRAF1 et LSP1 sont nécessaires pour une expression maximale du récepteur 4-1BB à la surface cellulaire de cellules B murines et coopèrent en aval de 4-1BB afin d'activer la cascade ERK dans les cellules B murines.

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at similar to3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 mug/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

Purification and characterisation of two cancer cachexia factors

Cachexia is a wasting phenomenon that often accompanies malignant disease. Its manifestation is associated with shortened survival and reduced responsiveness to anti-tumour therapy and as yet there is no established, effective amelioratory treatment. The MAC 16 model of cancer cachexia has been shown by many studies to closely mirror the human condition. Thus, cachexia is mediated by the presence of a small, slow-growing solid tumour that is mainly resistant to chemotherapy. In addition, the condition is largely attributable to aberrations in metabolic processes, while weight loss due to anorexia is negligible. Cachexia induced by the MAC 16 tumour, has been shown to be mediated by the production of tumour-derived circulatory catabolic factors, and the further elucidation of the structure of these molecules contributes towards the main content of this report. Thus, a factor with in vitro lipid-mobilising activity has been purified from the MAC 16 tumour, and has been found to have similarities to tumour-derived lipolytic factors published to date. Further work demonstrated that this factor was also purifiable from the urine of a patient with pancreatic cancer, and that it was capable of inducing weight loss in non tumour-bearing mice. Sequence analysis of the homogeneous material revealed an identity to Zn-α-2-glycoprotein, the significance of which is discussed. An additional factor, first detected as a result of its specific reactivity with a monoclonal antibody produced by fusion of splenocytes from MAC 16 tumour-bearing mice with mouse BALB/c myeloma cells, was identified as a co-purificant during studies to isolate the lipolytic factor. Subsequent purification of this material to homogeneity resulted in the determination of 18 of the N-terminal amino acids and revealed the highly glycosylated nature of its structure. Thus, this material (P24) was found to have an apparent molecular mass of 24kD of which 2kD was due to protein, while the remainder (92%) was due to the presence of carbohydrate groups. Sequence analysis of the protein core of P24 revealed an identity with Streptococcal pre-absorbing antigen (PA-Ag) in 11 of the amino acids, and the significance of this is discussed. P24 was shown to induce muscle protein breakdown in vitro and to induce cachexia in vivo, as measured by the depletion of fat (29%) and muscle (14%) tissue in the absence of a reduction of food and water intake. Further studies revealed that the same material was purifiable from the urine of patients with pancreatic cancer and was found to be detectable in the urine of cancer patients with weight loss greater than l.Skg/month. Thus, cachexia induced by the MAC 16 tumour in mice and by malignant disease in humans may be induced by similar mediators. Attempts to isolate the gene for P24 using information provided by the N-terminal protein sequence were unsuccessful. This was probably due to the low abundance o[ the material, as determined by protein purification studies; and the nature of the amino acids of the N-terminal sequence, which conferred a high degree o[ degeneracy to the oligonucleotides designed for the polymerase chain reaction.

Studies of the stability and metabolism of tumour inhibitory tetrazinones

Temozolomide is an imidazotetrazinone with antineoplastic properties. It is structurally related to dacarbazine. Temozolomide was not metabolized in vitro by liver fractions. Chemical decomposition appears to play an important r^ole in its in vitro and in vivo disposition. In contrast, 3-methylbenzotriazinone, a structural analogue, was metabolized by hepatic microsomes to afford benzotriazinone and a hydrophilic metabolite. The cytotoxicity of temozolomide, dacarbazine, 5-[3-(hydroxy-methyl-3-methyl-triazen-1-yl]imidazole-5-carboxamide (HMMTIC) and 3-monomethyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were investigated in TLX5 murine lymphoma cells. Unlike dacarbazine, which was not toxic, MTIC, HMMTIC and temozolomide were cytotoxic in the absence of microsomes. Decarbazine was only cytotoxic in the presence of microsomes. The formation of MTIC from dacarbazine, HMMTIC and temozolomide was determined by reversed phase high performance liquid chromatography in mixtures incubated under conditions identical to those described before. MTIC was generated chemically from temozolomide and HMMTIC metabolically from dacarbazine. Using [14C]temozolomide, it was found that, in mice, the major route of excretion of the drug is via the kidneys. An acidic metabolite (metabolite I) was found in the urine of mice which had received temozolomide but its identity has not been established. 1H NMR, UV and chemical analyses revealed that Metabolite I possesses an intact NNN linkage and the site of metabolism is at the N3 methyl group. A further acidic metabolite (metabolite II) was found in the urine of patients. Metabolite II was unambiguously identified as the 8-carboxylic acid derivative of temozolomide. In vitro cytotoxicity assay showed that ony metabolite II is cytotoxic but not metabolite I. Pharmacokinetic studies of temozolomide and MTIC in vivo were performed on mice bearing TLX5 tumour. Temozolomide was eliminated from the plasma monophasically with a t1/2 of 0.7hr. MTIC was identified as a product of decomposition. MTIC was eliminated rapidly with a t1/2 of 2min. Though temozolomide shares many biochemical and biological similarities with clinically used dacarbazine, the results obtained in this study show that it differs markedly in its pharmacokinetic properties from dacarbazine, as temozolomide produced relatively sustained plasma levels which were reflected by drug concentrations in the tumour.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.