(-)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1.

(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

CD47 agonist peptide PKHB1 induces immunogenic cell death in T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection.

UNLABELLED: During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE: Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm-a crowded environment where diffusion is slow-is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

Cuatro décadas de oncoinmunología experimental / Four decades of experimental oncoimmunology

En nuestro laboratorio, trabajando principalmente con ratones endocriados en un bioterio propio y adaptándonos a los sucesivos descubrimientos en cáncer experimental, se fueron añadiendo distintos subtemas, los cuales fueron surgiendo de los resultados obtenidos. Así fue que empezando con la inducción de leucemia en ratones por diversos procedimientos en vista a descubrir un virus leucemogénico humano, se llegó a estudiar el crecimiento tumoral y la relación tumor-huésped con especial énfasis en la resistencia concomitante. La comparación con la relación materno-fetal llevó al estudio de la tolerancia inmunológica en el recién nacido y de la inmunorregulación como base de la ontogenia del sistema inmune. Se contemplaron luego las alteraciones inmunológicas provocadas por estimulaciones antigénicas crónicas tanto en la madre como en el recién nacido, con el afán de encontrar una explicación al pico de leucemia observado en niños de poca edad. Estudiando los superantígenos asociados al MMTV (murine mammary tumor virus) se detectó una variante viral con deleción de determinados antígenos Vß del receptor T y fuerte inducción de tumores de mama en multipreñadas. Por otro lado, y por serendipismo, se descubrió que en ratones los progestágenos inducen cáncer de mama, dando origen a un modelo experimental que permite comparar el crecimiento neoplásico hormono-dependiente con el hormono-independiente o autónomo. De esta manera, después de tantos años, se reencuentran los subtemas tumorales y virales con los inmunológicos.

Cuatro décadas de oncoinmunología experimental / Four decades of experimental oncoimmunology

En nuestro laboratorio, trabajando principalmente con ratones endocriados en un bioterio propio y adaptándonos a los sucesivos descubrimientos en cáncer experimental, se fueron añadiendo distintos subtemas, los cuales fueron surgiendo de los resultados obtenidos. Así fue que empezando con la inducción de leucemia en ratones por diversos procedimientos en vista a descubrir un virus leucemogénico humano, se llegó a estudiar el crecimiento tumoral y la relación tumor-huésped con especial énfasis en la resistencia concomitante. La comparación con la relación materno-fetal llevó al estudio de la tolerancia inmunológica en el recién nacido y de la inmunorregulación como base de la ontogenia del sistema inmune. Se contemplaron luego las alteraciones inmunológicas provocadas por estimulaciones antigénicas crónicas tanto en la madre como en el recién nacido, con el afán de encontrar una explicación al pico de leucemia observado en niños de poca edad. Estudiando los superantígenos asociados al MMTV (murine mammary tumor virus) se detectó una variante viral con deleción de determinados antígenos Vß del receptor T y fuerte inducción de tumores de mama en multipreñadas. Por otro lado, y por serendipismo, se descubrió que en ratones los progestágenos inducen cáncer de mama, dando origen a un modelo experimental que permite comparar el crecimiento neoplásico hormono-dependiente con el hormono-independiente o autónomo. De esta manera, después de tantos años, se reencuentran los subtemas tumorales y virales con los inmunológicos. (AU)

Undersulfation of glycosaminoglycans reduces the proliferation of a leukemia cell line in vitro.

Leukemia represents the clonal expansion of an individual cell lineage of the hematopoietic system at a specific point of its maturation and development. This dysregulated expansion of cells is often accompanied by altered adherence to the bone marrow microenvironment and abnormalities in endogenous cytokine production by neoplastic cells. Proteoglycans (PGs) synthesized by neoplastic cells may interact with extracellular matrix (ECM) molecules and/or locally produced cytokines. It is believed that these events may be mediated by the glycosaminoglycan (GAG) moiety of PGs such as heparan or chondroitin sulfate, and depends on its charge. The strength of GAG-cytokine binding may be determined by the extent of sulfation of the GAG chains. The synthesis, metabolism and biological role of PGs in hematopoietic malignancies have not been clearly defined. In order to study how alterations of GAGs in leukemic cells may alter cellular behavior, we treated the murine myeloid leukemic cell line WeHi-3B with sodium chlorate. This drug reduces the sulfation of GAGs, since chlorate is a potent inhibitor of sulfate adenylyltransferase. The undersulfated GAGs produced by WeHi-3B cells were not efficient in controlling the mitotic rate of the cells, since a decrease in cell proliferation was observed in vitro. These data suggest that the complexes formed by GAGs with ECM components and/or cytokines may have an important role in the induction of leukemic cell proliferation. It is possible that the stimulatory activity elicited by this binding may be dependent upon the organization of these complexes.

Undersulfation of glycosaminoglycans reduces the proliferation of a leukemia cell line in vitro

Leukemia represents the clonal expansion of an individual cell lineage of the hematopoietic system at a specific point of its maturation and development. This dysregulated expansion of cells in often accompanied by altered adherence to the bone marrow microenvironment and abnormalities in endogenous cytokine production by neoplastic cells. Proteoglycans (PGs) synthesized by neoplastic cells may interact with extracellular matrix (ECM) molecules and/or locally produced cytokines. It is believed that these events may be mediated by the glycosaminoglycan (GAG) moiety of PGs such as heparan or chondroitin sulfate, and depends on its charge. The strength of GAG-cytokine binding may be determined by the extent to sulfation of the GAG chains. The synthesis, metabolism and biological role of PGs in hematopoietic malignancies have not been clearly defined. In order to study how alterations of GAGs in leukemic cells may alter cellular behavior, we treated the murine myeloid leukemic cell line WeHi-3B with sodium chlorate. This drug reduces the sulfation of GAGs, since chlorate is a potent inhibitor of sulfate adenylyltransferase. The undersulfated GAGs produced by WeHi-3B cells were not efficient in controlling the mitotic rat of the cells, since a decrease in cell proliferation was observed in vitro. These data suggest that the complexes formed by GAGs with ECM components and/or cytokines may have an important role in the induction of leukemic cell proliferation. It is possible that the stimulatory activity elicited by this binding may be dependent upon the organization of these complexes.

Caparratriene, an active sesquiterpene hydrocarbon from Ocotea caparrapi.

Caparratriene (1), a new sesquiterpene hydrocarbon with significant growth inhibitory activity (IC50 = 3.0 +/- 0.5 x 10(-6) M) against CEM leukemia cells, was isolated from the oil of Ocotea caparrapi (Nates) Dugand. The structure of 1, determined by spectroscopic techniques, corresponded to (E,E)-3,7,11-trimethyl-2,4,10-dodecatriene (C15H26).

Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor.

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

Age-related decline in cell-mediated immunity in the C58 leukemic mouse strain.

The leukemia-prone C58 strain of mouse was examined for age-related changes in cellular immune function. Proliferative responses of lymphocytes to autologous and allogeneic stimulator cells [autologous mixed lymphocyte response (AMLR) and mixed lymphocyte response (MLR), respectively] and to mitogens were tested both prior to and around the usual age of disease onset which occurs at 7-8 months. Leukemia in these animals was defined by elevated peripheral blood and splenic white blood cell counts. The AMLR declined greater than 30% by 6-7 months of age and was virtually absent by 8 months of age even in animals that were not overtly leukemic. The MLR declined precipitously (greater than 95%) at 9 months of age. Both declines occurred at a younger age in C58 mice than in nonleukemic strains. Mixing experiments with cells from young and old animals indicate a defect in the Ly 1+23-, L3T4+ responding T cells. No evidence indicating a role for suppressor cell activity in this decline of cell-mediated immunity could be found. Deficiencies in cytokine (IL-2 and IL-1) production were not observed except in the oldest mice tested. Around the usual time of disease onset, splenic natural killer (NK) cell activity declines sharply even in nonleukemic mice. Cell-mixing experiments showed no evidence of suppressor cell activity by spleen cells from older mice, leukemic or nonleukemic, on the NK cell activity of young adult animals. Interferon alpha, beta treatment enhanced the NK activity of cells from old mice but did not restore the level of activity seen in young mice. Evidence has therefore been found for a premature decline in cellular immune function in two responses with proposed immunoregulatory roles, the AMLR and NK cell activity. It is possible that their decline could play a predisposing role in the onset of this retroviral leukemia or that these cell populations may be the target of the retrovirus.

Two new quassinoids from Simaba multiflora fruits.

Two new quassinoids, 13, 18-dehydro-6 alpha-senecioyloxychaparrin (4) and 12-dehydro-6 alpha-senecioyloxychaparrin (5), have been isolated from Simaba multiflora fruits. Their structures were deduced from spectral data. 1H-13C 2-D chemical-shift correlation nmr was applied to the structural elucidation of the antileukemic quassinoid 4.

[Lack of relationship between viral leukemogenesis and immunosuppression in the mouse]. / Falta de relación entre la leucemogénesis viral y la inmunodepresión en el ratón.

The relationship between immunodepression and leukemogenesis induced by a murine retro-virus. PLLV-T2, was studied in different strains of mice. Cellular immunity, using as parameters both allograft rejection and graft versus host reaction, was not affected during the early phase of leukemia development. As for humoral immunity, determined by the response to xenogeneic red blood cells and to lipopolysaccharides, no direct relationship between the immunodepression caused by the retrovirus and leukemogenesis could be encountered: in certain strains, such as DBA/2, with susceptibility to leukemogenesis similar to that of BALB/c, no decrease in the immune response was registered during the early phase of the disease. The results obtained demonstrate that immunodepression is not a necessary condition for the clinical appearance of this viral-induced leukemia indicating that humoral and/or cellular immunity would not play an important role as surveillance mechanism against this neoplasia.

Falta de relacion entre la leucemogenesis viral y la inmunodepresion en el raton. / Lack of relation between viral leukomogenesis and immunodepression in mice

Se estudio la relacion entre la inmunodepresion y la leucemogenesis causadas por un retrovirus murino, el PLLV-T2, en distintas cepas de ratones. Se demostro que la inmunidad celular, medida por la reaccion de rechazo de tejido alogenico y por la reaccion de injerto contra huespede, no fue afectada por el virus en una etapa temprana del desarrollo leucemico.Con respecto a la inmunidad humoral aun cuando PLLV-T2 producia una marcada inmunodepresion temprana en algunas cepas de ratones, se demostro que este fenomeno no era universal porque no solo no habia una relacion directamente proporcional entre susceptibilidad a la oncogenesis e inmunodepresion sino que habia algunas cepas, como la DBA/2, que teniendo una susceptibilidad a los efectos leucemogenos del virus similar a los de la cepa BALB/c, no mostraban una respuesta humoral disminuida en la primera fase de enfermedad. Los datos de este trabajo muestran que un fenomeno de inmunodepresion so seria un prerrequisito para la manifestacion clinica de la leucemia inducida por PLLV-T2, indicando que ni la respuesta humoral ni la celular actuarian como um mecanismmo de vigilancia inmunologica contra esta neoplasia