Leukemia treatment: studies exploring bone marrow microenvironment, drug resistance and cannabidiol

The results described in this thesis highlight the role of human regenerative associated endothelial cells (hRECs) in the bone marrow microenvironment and hematopoiesis. By being resistant to 5-FU therapy, hRECs remain active in secreting factors enabling enhanced expansion of precursor HSC. A second part of this thesis provides novel insights into the role of specific influx and efflux transporters implicated in the efficacy/resistance of MTX and Ara-C. Finally, a third part described the results of in vitro experimental anti-leukemia therapeutics with cannabidiol, revealing double-edged effects: anti-leukemic effects at high doses of CBD and cell growth stimulatory effects at low doses of CBD. Together, these results may contribute to improve anti-leukemic chemotherapy efficacy and minimize toxicity

Genetic variants of d4T drug transporters and dNTP pool regulators, and their association with response to d4T-ART

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Genetics. Johannesburg, South Africa 2017Background: Stavudine (d4T) use is associated with the development of sensory neuropathy (SN), several mechanisms may underlie d4T-induced toxicity, including: (1) Inter-patient genetic variability in the genes modulating the deoxynucleotide triphosphate (dNTP) pool sizes. (2) Variation in intracellular ARV drug concentrations due to genetic variation in drug transporters. In our study we examined the genetic variation in four stavudine transporter genes and seven genes regulating the deoxythymidine triphosphate (dTTP) synthesis and their associations with d4T-induced SN or CD4+ T cell count or mtDNA copy number. Methods: We examined a cohort of HIV-positive South African (SA) adults exposed to d4T, including 143 cases with SN and 120 controls without SN. 26 single nucleotide polymorphisms (SNPs) from the literature were chosen, prioritised on being tagSNPs with minor allele frequency >5% in Kenyan Luhya (a proxy population for the SA Black population); SNP functional effects and suitability for multiplex analysis on the genotyping platform. Genotyping was performed using Sequenom mass spectrometry. A qPCR assay was used to measure the mtDNA copy number. Association of sensory neuropathy, CD4+ T cell count and mtDNA copy number with genetic variants was evaluated using PLINK. Results: All 26 SNPs were in Hardy-Weinberg equilibrium (HWE) in both the cases and controls. SNP rs8187758 of the SLC28A1 transporter gene and a 3-SNP haplotype ABCG2 were significantly associated with CD4+ T cell count after correction for multiple testing (p = 0.043 and p=0.042 respectively), but were not significant in multivariate testing. No SNP remained significantly associated with SN or mtDNA copy number, after correction for multiple testing. Conclusion: Variation in genes encoding molecular transporters of d4T may influence CD4+ T cell counts after ART. This study presents a positive step towards achieving personalized medicine in SA.MT 201

siRNA Targeting of Thymidylate Synthase, Thymidine Kinase 1 and Thymidine Kinase 2 as an Anticancer Therapy: A Combinatorial RNAi Approach

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS siRNA to sensitize tumour cells to TS-targeting drugs. Downregulation of TKs with siRNA enhanced the capacity of TS siRNA to sensitize tumour cells to traditional TS protein-targeting drugs (5FUdR and pemetrexed). Combined downregulation of these enzymes is an attractive strategy to enhance TS-targeted anticancer therapy. TK2 can phosphorylate both thymidine and deoxycytidine to generate dTMP and dCMP, precursors for dTTP and dCTP, respectively. dCTP negatively regulates deoxycytidine kinase (dCK), another enzyme that phosphorylates deoxycytidine as well as the anticancer drug gemcitabine. Antisense knockdown of TK2 could reduce TK2-produced dCMP, thus decreasing dCTP levels and inhibition of dCK, and lead to increased dCK activity, gemcitabine activation, and anticancer effectiveness. Given the substrate promiscuity of TK2, we hypothesized that: (1) TK2 can mediate human tumour cell resistance to gemcitabine, (2) antisense downregulation of TK2 can overcome that resistance, and (3) TK2 siRNA-induced drug sensitization results in mitochondrial damage. siRNA downregulation of TK2 expression sensitized MCF7 and HeLa cells to gemcitabine, but did not sensitize A549 cells (low TK2 expresser). Treatment with TK2 siRNA and gemcitabine: 1) decreased mitochondrial redox status, 2) decreased mitochondrial DNA (mtDNA:nDNA ratio), and 3) decreased mitochondrial activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 from other dTMP-producing enzymes

Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides

Mutagenesis Studies of the Varicella-Zoster Virus Thymidylate Synthase

Varicella-zoster virus (VZV), the causative agent of chickenpox and shingles, encodes approximately 70 different proteins including the enzyme thymidylate synthase (TS). TS catalyses the reductive methylation of deoxyuridylate to form thymidylate using N5,N10-methylene-tetrahydrofolate as a cofactor. The enzyme plays a pivotal role in the provision of an essential precursor for DNA synthesis. As such it has been used as a target for antimetabolites which are effective against a variety of infectious and proliferative diseases. The work presented in this thesis describes the establishment of two random mutagenesis protocols suitable for the generation of complete mutant DNA libraries of the VZV TS gene. Use of these libraries, in conjunction with a novel screening protocol, has shown that a single nucleotide change is not sufficient to give rise to a VZV TS variant resistant to the potent inhibitors 5-fluoro-2'-deoxyuridine-monophosphate (FdUMP) and 5-ethynyl-2'-deoxy-uridine-monophosphate (EYdUMP). This is in contrast to a variant of human TS which contains a single nucleotide substitution that has been proposed to confer resistance to FdUMP. The introduction of the equivalent nucleotide substitution into the VZV TS gene by site-directed mutagenesis (which resulted in the equivalent amino acid change) did not result in a drug resistant viral enzyme. The mutant library was used to isolate variants of VZV TS that lack catalytic activity. Molecular modelling of the effects of the amino acid substitutions that gave rise to these inactive variants, extended our understanding of the plasticity of protein structure. As a complementary approach to investigating the structure and function of TS, site-directed mutagenesis was used to study the effects of amino acid changes in two specific regions of the protein; one buried and one exposed to solvent. In the buried region it was shown that the enzyme is more flexible to amino acid substitutions than previously expected, and the mechanism of accomodation of at least one of the changes was identified. A study of the solvent exposed C-terminal region identified structural and functional differences between VZV TS and Lactobacillus casei TS. The two enzymes responded differently to amino acid changes, and the VZV enzyme relied more heavily on hydrophobic contacts to maintain a functional conformation. Phylogenetic comparisons and molecular modelling suggested that the VZV TS may be unique in this regard. Exploitation of the difference between the VZV TS and the human enzyme may allow the rational design of VZV-specific inhibitors and such compounds may have a role as anti-varicella drugs. It was shown that the expression of the active VZV thymidine kinase (TK) was necessary for the phosphorylation of 5-ethynyl-2'-deoxyuridine to a form that would inhibit TS. This led to the identification of an amino acid essential for VZV TK activity in a region of the enzyme not previously thought to play a role in catalysis. The evolutionary source of the herpesvirus TKs has been hitherto obscure. A comparison of the conserved amino acid regions of herpesvirus TKs with cellular deoxycytidine kinase revealed a significant degree of homology. In addition, the herpesvirus TKs and deoxycytidine kinase share common biochemical properties. Taken together, this led to the proposal that the evolutionary source of the herpesvirus TKs was the cellular deoxycytidine kinase

Dissecting SAMHD1´s role in the type I Interferon induced early block to HIV-1 infection and its connection to cancer

Human immunodeficiency virus-1 (HIV-1) and all other viruses are known to interact with multiple host cellular proteins during their replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to repress viral replication. These host cellular proteins are known as restriction factors and they represent the host's first line of defense against the viral pathogens. Sterile alpha motif and HD domain containing 1 (SAMHD1) has been identified as a HIV-1 restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells. SAMHD1 is the target of the viral protein x (Vpx) from simian lentiviruses and HIV-2. Vpx mediates the recruitment of the Cullin4-DDB1-DCAF1 ubiquitin ligase machinery to SAMHD1 leading to polyubiquitination and subsequent degradation of SAMHD1. Previous studies on monocyte-derived dendritic cells suggested that the Vpx-induced rescue of HIV-1 infection from early type I IFN-induced blocks was independent of SAMHD1, since Vpx mutant Q76A, which is unable to recruit DCAF1 and to degrade SAMHD1, still increased HIV-1 infection in type I IFN treated cells. The rescue in healthy blood donor cells was not observed when Q76A mutant Vpx virus-like particles were used, suggesting that – in conflict with previous reports – SAMHD1 degradation is required for efficient Vpx-mediated rescue of HIV-1 from the type I IFN-induced early antiviral blocks. To investigate the role of SAMHD1 in the Vpx-mediated rescue of HIV-1 from the type I IFN-induced block in myeloid cells at more detail, we generated CRISPR/Cas9 THP-1 cells, a monocytic acute myeloid leukemia cell line, lacking a functional SAMHD1 gene. In line with previous studies, the lack of SAMHD1 protein had no impact on the level of the type I IFN-induced early block to HIV-1 infection as compared to control or parental THP-1 cells. However, while Vpx was able to rescue HIV-1 infectivity in parental THP-1 or CRISPR/Cas9 control cells from the type I IFN effects, no rescue was observed when SAMHD1 protein was absent. To investigate whether the enzymatic activity of SAMHD1 was required for the Vpx-mediated rescue of HIV-1 infection from the early type I IFN-induced blocks, we reconstituted expression of wild type or different catalytically-inactive SAMHD1 mutants in SAMHD1-/- cells and found that Vpx increased HIV-1 infectivity in the presence of wild type, but not H233A mutant SAMHD1, suggesting that the enzymatic activity of SAMHD1 is required for a Vpx-induced rescue of HIV-1 infection from the type I IFN-induced block. We also generated a CRISPR/Cas9 THP-1 cell clone, which had one disrupted SAMHD1 allele and one allele, in which the entire nuclear localization signal (11KRPR14) was deleted in frame, generating an internally NLS-disrupted endogenously expressed SAMHD1 protein. In these cells, SAMHD1 was predominantly localized to the cytoplasm, although a 5 fraction was also observed in the nucleoplasm, suggesting for an alternative nuclear import pathway, independent of the classical 11KRPR14 NLS. In these cells, Vpx still rescued HIV-1 from the type I IFN-induced early block to infection. Of note, SAMHD1 degradation was profoundly delayed, suggesting that Vpx-induced polyubiquitination of SAMHD1 is sufficient to overcome the early IFN-induced block to HIV-1 in myeloid cells. SAMHD1 not only acts as a host restriction factor against lentiviral, endogenous retroviruses, hepatitis B virus, herpesviruses (HSV-1) and poxviruses, mutations in the SAMHD1 gene have also been linked to the immune disorder Aicardi-Goutières Syndrome (AGS), a genetic disease mimicking congenital virus infection. Recurrent mutations and reduced expression levels of SAMHD1 have been suggested to play a role for the oncogenesis of several cancers such as colon and Rectum Adenocarcinoma (COAD/READ), lung cancer, cutaneous T-cell lymphoma, acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL). Interestingly, SAMHD1’s function as a possible tumor suppressor is complexed by its role as a resistance factor in nucleoside analogue-based anti-cancer therapies. Cytarabine (ara-C), a deoxycytidine analog, is the single most important drug in the treatment of AML and other hematological malignancies, exerting its cytotoxic effects through its activated triphosphate (ara-CTP), eventually leading to DNA damage and cell death. We and others demonstrated that SAMHD1 is able to detoxify cells by hydrolytic activity towards ara-CTP. Accordingly, primary AML blasts treated with Vpx to deplete SAMHD1 as well as THP-1 CRISPR/Cas9 SAMHD1 knock-out cells showed increased sensitivity towards ara-C induced cytotoxicity. Using these knock-out cells as a back-bone, we expressed a large panel of SAMHD1 mutants harboring non-synonymous single nucleotide polymorphisms (SNPs) that have been identified in patients with AML, READ, STAD or COAD and performed differential analyses of ara-C sensitivity as well as restriction activity towards HIV-1 infection to unravel possible mechanistic differences in both activities. In this respect neutralization of ara-C induced cytotoxicity was found to be a very good surrogate for the enzymatic dNTPase activity of SAMHD1, and using naturally occurring SAMHD1 variants ensured that these proteins were not artificially defective, hence for the first time a direct comparison of enzymatic activity and anti-HIV-1 activity could be investigated in the same cells. We are currently investigating the effects of these SAMHD1 SNPs on oligomerization and sensitivity for degradation by Vpx. The identification of SAMHD1 SNPs altering the sensitivity to certain anti-cancer chemotherapies could also be a key for future personalized treatment strategies. Furthermore, the ability of our assays to uncouple SAMHD1 enzymatic activity from virus restriction could help to understand the contribution of SAMHD1’s dNTPase activity towards HIV-1 restriction and since the Vpx-induced rescue of HIV-1 infection from the type I IFN induced block was shown to depend on SAMHD1, may help to unravel the IFN-induced early blocks against HIV-1. In Summary, SAMHD1 plays a bigger role in the type I IFN-induced block than currently is appreciated and further investigation of its cellular function may provide insights into the underlying mechanisms and contributing additional factors

Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.Vita.Includes bibliographical references.Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield.(cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported.(cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP.(cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP.by Gregory J.S. Lohman.Ph.D

Novel approaches to understanding Hent2 and hENT2-related proteins: From novelnuclear variants to global networks / Nuevos enfoques sobre el estudio de hENT2 y proteínas relacionadas: desde nuevas variantes nucleares a redes globales

hENT2 is the human nucleoside transporter for which most splice variants have been described to date, in contrast to hENT1 for which no variants have been reported yet. In this dissertation, we have identified 3 novel splice variants of hENT2 which encode 2 novel nuclear isoforms of this transporter protein. As we demonstrated, these variants appear to be functional and ubiquitously expressed among cell lines and tissues, although the pattern of expression levels may vary depending on the growth conditions and the cellular needs. The physiological role of these novel nuclear variants of hENT2 still remains unclear, since there is no evidence of the presence of the nucleoside salvage pathway inside the nucleus. Therefore, the presence of nucleoside transporters at the nuclear membrane might not have any relevant meaning. Nonetheless, based on the protein-protein interactions obtained from the screening with MYTH technique, other functional implications are coming up. These nuclear variants of hENT2 could be involved in alternative splicing regulation, since they appeared to interact with several splicing factors. As we proved, hENT2 splicing patterns vary depending upon growth conditions, suggesting the existence of a regulation (or even a self-regulation) mechanism of the splicing phenomenon. Several proteins have been previously described as self-regulators of alternative splicing (Sureau et al. 2001; Sun et al. 2010; de Morree et al. 2012). In this dissertation we propose the hypothesis of HNP32 and HNP36 proteins, localized at the nuclear membrane, interacting with splice regulation factors and modulating their availability to bind pre-mRNA, thus determining the splice sites recognized by the spliceosome. In such a way, hENT2 itself would regulate the ratio between the wt isoform and the nuclear variants to adapt itself to the cellular situation. According to our in vitro phosphorylation assays, hENT2 could be phosphorylated by CKII inside the cell. CKII is a kinase that appears to be unregulated because it is constitutively active. Therefore, it has been suggested that changes in the phosphorylation status of CKII substrates would depend on regulated dephosphorylation (Pinna 1990). Considering the putative interaction between hENT2 and PP1, we propose a possible regulation of hENT2 by phosphorylation, where the transporter would be phosphorylated by default and regulation would come by dephosphorylation via PP1. Actually, considering our biotinylation results where we found a double band at 54 KDa, the supposed phosphorylated protein would remain intracellular while the supposed non phosphorylated protein would be active at the plasma membrane. Although we could not demonstrate a direct in vitro phosphorylation of hENT2 by PKC, this kinase appears to be involved in hENT2 trafficking regulation. Activation of PKC promotes hENT2 trafficking to the plasma membrane although it does not necessarily entails an increase of hENT2-related transport activity. Actually, the clear consequence of the hENT2 translocation to the plasma membrane is a significant decrease on hENT1 activity. We propose the theory of a dual population of hENT2 proteins at the plasma membrane. One non-phosphorylated hENT2 isoform of 54 KDa could be related with transport activity, while another isoform of 45 KDa would not be active as a transporter, despite being present at the plasma membrane. Similarly, two different kinds of regulation by phosphorylation would affect hENT2 function, either regulating its trafficking to the plasma membrane via PKC, or activating its function as a nucleoside transporter via PP1. These two putative pathways of regulation would not necessarily be independent of each other. Finally, our transcriptomic analysis of gastro-hepatic cell lines allowed us to place ENT2 within a global gene network involved in cell proliferation and survival. PKC appeared to be a possible central point of regulation of that network, coordinating a global response of the cell to the growth and environmental conditions. In addition, part of that novel network, including hENT2, could be involved in response to paclitaxel treatment. As we understand, a positive response to the therapy would not be determined by the hENT2 role as a transporter, but by the context of this network connecting several genes involved in proliferation and cell survival.La bioasequibilidad y la acción de los fármacos derivados de nucleósidos utilizados en la terapia antitumoral dependen en gran medida de la capacidad de las células diana para internalizarlos y activarlos metabólicamente. Estos fármacos interfieren en los procesos de captación y metabolización de los nucleósidos naturales, incidiendo así en una de las rutas metabólicas probablemente mejor conservadas evolutivamente, la vía de recuperación, finamente regulada y de notable eficacia energética si se tiene en cuenta el coste asociado a la síntesis de novo de nucleósidos (Molina-Arcas et al. 2006). Dado su carácter hidrofílico, tanto los nucleósidos como sus derivados requieren de transportadores de membrana para poder ser internalizados. En la presente tesis, hemos identificado 3 nuevas variantes de splicing del transportador de nucleósidos equilibrativo 2 (hENT2), las cuales codifican dos nuevas isoformas nucleares de la proteína. Dichas variantes parecen ser funcionales y estar ubicuamente expresadas en líneas celulares y tejidos sanos. Se han realizado estudios de regulación por fosforilación del transportador hENT2 y hemos demostrado que hENT2 es fosforilado in vitro por la kinasa CKII. Dado que CKII se encuentra constitutivamente activa, hENT2 podría estar regulado por desfosforilación a través de PP1. Además, la activación de PKC mediante el tratamiento con PDD promueve la translocación de de hENT2 hacia la membrana plasmática en células HEK-293, a la vez que disminuye la actividad transportadora de hENT1. En base a nuestros resultados proponemos un modelo en que hENT2 podría tener una población dual en la membrana plasmática. De manera que una isoforma no fosforilada de hENT2, de unos 54 KDa, podría estar relacionada con la actividad transportadora, mientras que otra isoforma de 45 KDa no sería activa como transportador, a pesar de estar situada en la membrana plasmática. Por otro lado, estudios de faramocegónimca nos han permitido definir una red global de genes aparentemente envueltos en proliferación celular y proliferación. ENT1, ENT2 y CNT3 forman parte de esta red génica, en la cual PKC podría tener un papel central regulador. ENT2, junto con otros genes implicados en la proliferación celular y supervivencia, correlacionan negativamente con la supervivencia celular en respuesta al tratamiento con paclitaxel en líneas celulares gastro-hepáticas