Anaplastic large cell lymphoma-propagating cells are detectable by side population analysis and possess an expression profile reflective of a primitive origin.

Cancer stem cells or tumour-propagating cells (TPCs) have been identified for a number of cancers, but data pertaining to their existence in lymphoma so far remain elusive. We show for the first time that a small subset of cells purified from human anaplastic lymphoma kinase (ALK)-positive and -negative, anaplastic large cell lymphoma cell lines and primary patient tumours using the side population (SP) technique have serial tumour-propagating capacity both in vitro and in vivo; they give rise to both themselves and the bulk tumour population as well as supporting growth of the latter through the production of soluble factors. In vivo serial dilution assays utilising a variety of model systems inclusive of human cell lines, primary human tumours and nucleophosmin (NPM)-ALK-induced murine tumours demonstrate the TPC frequency to vary from as many as 1/54 to 1/1336 tumour cells. In addition, the SP cells express higher levels of pluripotency-associated transcription factors and are enriched for a gene expression profile consistent with early thymic progenitors. Finally, our data show that the SP cells express higher levels of the NPM-ALK oncogene and are sensitive to an ALK inhibitor.

Dominant-negative Ikaros cooperates with BCR-ABL1 to induce human acute myeloid leukemia in xenografts.

Historically, our understanding of mechanisms underlying human leukemogenesis are inferred from genetically engineered mouse models. Relatively, few models that use primary human cells recapitulate the full leukemic transformation as assayed in xenografts and myeloid transformation is infrequent. We report a humanized experimental leukemia model where xenografts develop aggressive acute myeloid leukemia (AML) with disseminated myeloid sarcomas within 4 weeks following transplantation of cord blood transduced with vectors expressing BCR-ABL1 and a dominant-negative isoform of IKAROS, Ik6. Ik6 induced transcriptional programs in BCR-ABL1-transduced progenitors that contained repressed B-cell progenitor programs, along with strong stemness, proliferation and granulocyte-monocytic progenitor (GMP) signatures-a novel combination not induced in control groups. Thus, wild-type IKAROS restrains stemness properties and has tumor suppressor activity in BCR-ABL1-initiated leukemia. Although IKAROS mutations/deletions are common in lymphoid transformation, they are found also at low frequency in AML that progress from a prior myeloproliferative neoplasm (MPN) state. Our experimental system provides an excellent model to gain insight into these rare cases of AML transformation and the properties conferred by IKAROS loss of function as a secondary mutation. More generally, our data points to the importance of deregulated stemness/lineage commitment programs in human myeloid leukemogenesis.

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease.

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy.

An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.

Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach.

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.

Can estrogenic radicals, generated by lactoperoxidase, be involved in the molecular mechanism of breast carcinogenesis?

Mutations of regulatory genes, which perturb the mechanism of cell replication resulting in abnormal cell proliferation, are the main cause of cancer. Many endogenous and exogenous chemicals (including estrogenic hormones) are known to represent a major carcinogenic risk for humans. 2-OH- and 4-OH-derivatives of estrogenic molecules have been shown to form stable adducts with purine DNA bases and act as 'depurinating' agents, thus altering gene transcription (Cavalieri EL, Stack DE, Devanesan PD et al. Proc Natl Acad Sci USA 1997; 94: 10937-10942). Lactoperoxidase (LPO), which is produced by mammary glands, is likely to be involved in breast carcinogenesis, because of its ability to interact with estrogenic hormones and oxidise them through two one-electron reaction steps. We investigated the reactivity of LPO towards five molecules: 17-beta-estradiol (a natural hormone), diethylstilbestrol (a synthetic drug, supplied to pregnant women for preventing spontaneous abortion), exestrol (a synthetic antigonadotropic estrogen), 2-OH- and 4-OH-estradiol (catabolic products of estradiol). Enzymatically generated radical derivatives of such molecules were stabilized by spin-trapping or by chelation of a diamagnetic metal ion and characterized with EPR spectroscopy. A kinetic study of the oxidation process was carried out using EPR and UV-visible spectroscopy.

Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase.

The well-known and easily available horseradish peroxidase (HRP) catalyzes the H2O2-dependent oxidative 4-dechlorination of the pollutant 2,4,6-trichlorophenol, which is recalcitrant to many organisms except those producing ligninases. UV-visible spectroscopy and gas chromatography-mass spectrometry identified the oxidized reaction product as 2,6-dichloro-1,4-benzoquinone. NMR and IR spectroscopic data further supported the above characterization. Experimental evidence for the elimination of HCl from the substrate was acquired by detecting the decrease in pH of the reaction mixture, and by observing the presence of the beta-chlorocyclopentadienone cation fragment in the mass spectrum of 2,6-dichloro-1,4-benzoquinone. Consequently, nucleophilic attack by water on the 2,4,6-trichlorocyclohexadienone cation was proposed to give the final product. Our results indicate an oxidative dechlorination pathway catalyzed by HRP for 2,4,6-trichlorophenol, similar to that by extracellular lignin peroxidases. The relative catalytic efficiency of HRP seems higher than that of lignin peroxidases. The HRP-H2O2 catalytic system could be utilized in the degradation of polychlorinated phenols for industrial and biotechnological purposes.

Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase.

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.

Spectroscopic investigations on the highly purified lactoperoxidase Fe(III)-heme catalytic site.

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techniques and image processing. A detailed report is given of the experimental procedure. Furthermore, electron paramagnetic resonance has played a fundamental role in evaluating the enzyme purity against lactoferrin and minor LPO isoenzyme components in setting the final steps of the purification. With the aim to completely clarify the Fe(III)-heme high-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 (RZ = 0.95) from farm and commercial milk, respectively, were purified and characterized in particular by electron paramagnetic resonance (EPR) spectroscopy, in comparison with a commercial preparation (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indictive of the presence of an iron(III)-heme high-spin catalytic site in the native enzyme. On the contrary, in the LPO2 spectrum a thermal equilibrium between high- and low-spin iron(III)-heme species is present. The low-spin component of the spectrum has been assigned to an LPO-NO2- adduct due to the presence of some nitrite impurities originating either from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shwos the presence of some spurious lines in the g approximately equal to 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously reported in the literature contain a variable number of spurious lines in the g approximately equal to 4 and 2 regions as a consequence of lactoferrin impurity and LPO low-spin adducts with endogenous or exogenous anions. Furthermore, the interaction of LPO with its native substrate (the thiocyanate anion), which previously was shown by NMR and EPR (at high substrate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscopy at room temperature and high substrate concentration as a function of pH. The LPO activity at optimum pH (approximately equal to 4-5) has been measured in phosphate and acetate buffer using as an oxidizable substrate the system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (DMAB-MBTH), which was considered a good chromogen for other peroxidases such as HRP and zucchini peroxidases. The LPO vs SCN- activity at optimum pH (approximately equal to 5.5) has been measured in phosphate and acetate buffer.(ABSTRACT TRUNCATED AT 400 WORDS)