Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.     

Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family.

Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N-glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome.     

Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.     

Astroglial cytoprotection by erythropoietin pre-conditioning: implications for ischemic and degenerative CNS disorders

Erythropoietin (Epo) is a glycoprotein secreted by the kidney in response to hypoxia that stimulates erythropoiesis through interaction with cell surface Epo receptors. Pre-treatment with Epo has been shown to protect neurons in models of ischemic injury. The mechanism responsible for this neuroprotection and the effects of Epo on astroglial and other non-neuronal cell populations remain unknown. In the present study, we determined whether Epo pre-treatment protects neonatal rat astrocytes from apoptotic cell death resulting from treatment with nitric oxide, staurosporine (STS) and arsenic trioxide and possible mechanisms mediating Epo-related cytoprotection. Epo (5-20 U/mL) significantly attenuated multiple hallmarks of apoptotic cell death in astroglia exposed to nitric oxide and STS but not arsenic trioxide. Epo (20 U/mL) induced mild oxidative stress as shown by increases in heme oxygenase (HO)-1 mRNA and protein expression that could be suppressed by antioxidant coadministration. Moreover, coincubation with tin-mesoporphyrin, a competitive inhibitor of HO activity, abrogated the cytoprotective effects of Epo (20 U/mL) in the face of STS treatment. Thus, induction of the ho-1 gene may contribute to the glioprotection accruing from high-dose Epo exposure. Epo may augment astroglial resistance to certain chemical stressors by oxidative stress-dependent and -independent mechanisms.     

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.     

Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest

Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.     

Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.

We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.     

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.Despite good remission rates observed in acute myeloid leukemia (AML) patients, the 5-year event-free survival rates reach only 35–40% in adults and 60–70% in children (1, 2). Apoptosis is one of the crucial mechanisms influencing survival of AML cells, and its deregulation can possibly lead to chemotherapy resistance and eventually relapse (3–5). The ability of cells to undergo apoptosis is largely defined by the relative expression of anti- (i.e. BCL-2, BCL-X long isoform - BCL-XL, or MCL-1) and proapoptotic (i.e. BAX, BH3 interacting domain death agonist - BID, caspases) proteins. Several studies have shown that the levels of BCL-2 and BCL-2/BAX ratio are a determinant of apoptosis-resistance in AML blasts and are associated with survival in AML patients (3, 6). We have previously demonstrated that the expression of several apoptosis-related proteins, such as BCL-2, BCL-XL, MCL-1, and BAX, can be reliably measured in AML samples by flow cytometry (6). These four quantitative parameters, which constitute an anti-apoptosis index (AAI)¹, have proven to be a reliable predictor of AML patients'' survival, with a high apoptosis-resistant profile (i.e. higher AAI) of diagnosis leukemic blasts being associated with shorter disease-free survival (7). Accordingly, AAI determined at the time of diagnosis also correlated with the frequency of minimal residual disease (MRD), which is a reflection of drug-resistant leukemic cells that have survived chemotherapy (7). MRD can be detected at a low frequency in bone marrow (BM) at the time of remission and is thought to contain the relapse-initiating cells (8–10). These observations imply that leukemic cells that harbor an apoptosis-resistant protein profile at diagnosis can better survive chemotherapy, thereby eventually causing a relapse. Consequently, we further hypothesized that the AAI of MRD cells would be either elevated or at least similar to the profile of leukemic cells at diagnosis. Surprisingly, in complete remission patients, the AAI decreased in the MRD situation compared with apoptosis-resistant profile as measured in leukemic blasts at diagnosis. The values of the AAI profile increased again at relapse, indicating apoptosis-resistance (11). Based on these unexpected findings, we hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly also in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment.Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer. In this respect, both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment (12–14). Apoptosis in the AML setting can be regulated by several cytokines as well as by EVs, which carry variable cargoes, including multiple proteins (15–18). In line with our hypothesis, apoptosis of BM cells was shown to be inhibited in the presence of secretome derived from AML blasts (19). These observations suggest that factors secreted by apoptosis-resistant leukemic blasts are likely to confer a drug resistance phenotype upon initially sensitive blasts. Therefore, the aim of our current study was to characterize the microenvironment produced by apoptosis-resistant AML blasts in terms of its capacity to influence apoptosis regulation in neighboring cells and protein content.     

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.