RESUMO
The SARS-CoV-2 (COVID-19) pandemic is a worldwide public health emergency with widespread impact on health care delivery. Unforeseen challenges have been noted during administration of usual haematology care in these unusual COVID-19 times. Medical services have been overstretched and frontline health workers have borne the brunt of COVID-19 pandemic. Movement restrictions during lockdown prevented large sections of population from accessing health care, blood banks from holding blood drives, and disrupted delivery of diagnostic hematology services. The disruption in hematology care due to COVID-19 pandemic in India has been disproportionately higher compared to other subspecialities as hematology practice in India remains restricted to major cities. In this review we chronicle the challenges encountered in caring for hematology patients during the COVID-19 pandemic in India and put forth recommendations for minimizing their impact on provision of hematology care with special emphasis on hematology practice in lower and middle income countries (LMICs).
RESUMO
Umbilical cord blood (UCB) is a powerful storehouse for normal CD34+ haematopoietic stem cells (HSCs), often used for allogeneic bone marrow (BM) transplantation in malignant and non-malignant diseases. The glycomic especially the sialoglycomic aspect of these HSCs has been unravelled in this study. Cell surface expression of the glycans with the related enzymatic activities has been compared with the BM of childhood acute lymphoblastic leukaemia, a common BM-associated malignancy. An enhanced cell surface expression of α2,3-linked sialic acid, P- and E-selectins, and intercellular adhesion molecule along with reduced expression of L-selectin distinguishes CD34+ HSCs of UCB from leukaemic samples. More importantly, high expression of O-acetylated sialoglycoproteins, a hallmark of lymphoblasts, is drastically reduced in the CD34+ HSCs of UCB and is substantiated by the low activity of sialylate-O-acetyltransferase and high sialidase activity. In contrast, a significant variation is evident in the expression of sialic acid, α2,6-linked sialic acids, and the sialyltransferase activity. Taken together, these studies indicate a few signature molecules, forming a unique glycomic template, which may be a potential indicator, reassuring the normal profile of these stem cells, to be used for future transplantation.
Assuntos
Sangue Fetal/citologia , Glicômica , Células-Tronco Hematopoéticas/química , Antígenos CD34 , Células-Tronco Hematopoéticas/citologia , Humanos , Linfócitos , Ácidos Siálicos/químicaRESUMO
Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH(+)SSC(lo)CD45(hi)Neu5,9Ac2 -GPs(lo)CD34(+)CD38(-)CD90(+)CD117(+)CD133(+)) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH(+)SSC(lo)CD45(lo)Neu5,9Ac2 -GPs(hi)CD34(+)CD38(+)CD90(-)CD117(-)CD133(-)) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.
Assuntos
Aldeído Desidrogenase/metabolismo , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ácidos Siálicos/metabolismo , Acetilação , Adolescente , Diferenciação Celular , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Células-Tronco Neoplásicas/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologiaRESUMO
Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4(+) CD25(+) FoxP3(+) Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals (n = 35). These diagnosed patients demonstrated a lower number of CD4(+) CD25(+) cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-ß and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4(+) CD25(-) responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4(+) CD25(+) FoxP3(+) Treg cells became high with the progression of malignancy in B-ALL. Relative distribution of Tresp and Treg cells was only ~5 : 1 in B-ALL but ~35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4(+) CD25(+) cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4(+) CD25(+) FoxP3(+) Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL.
Assuntos
Biomarcadores Tumorais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Citocinas/metabolismo , Progressão da Doença , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Indução de Remissão , Linfócitos T Reguladores/efeitos dos fármacos , Resultado do TratamentoRESUMO
In search of genetic alterations responsible for high fetal hemoglobin (Hb F) phenotypes in the population of eastern India, 91 probands were screened for four polymorphisms by sequencing and/or restriction fragment length polymorphism (RFLP) analysis. These are the A>G allele on the rs4895441 locus in the intergenic region between HBS1L and MYB on chromosome 6, the G>A allele on the rs4671393 locus on chromosome 2 (BCL11A gene), the A>C allele on the rs2071348 (HBBP1 gene) and the XmnI polymorphism (rs7482144, -158 position of HBG2) on chromosome 11. We found a significant association (p = 0.002 and 0.0013) of Hb F levels with rs2071348 and rs4895441, respectively. However, the polymorphism rs4671393 gene did not show significant association with Hb F levels (p = 0.0655). As is well known, the XmnI polymorphism (p <0.0001) showed the strongest association.
Assuntos
Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Genes myb , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , gama-Globinas/genética , Adolescente , Adulto , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Criança , Pré-Escolar , DNA Intergênico , Feminino , Humanos , Índia , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Polimorfismo Genético , Proteínas Repressoras , Adulto Jovem , Globinas beta/genéticaRESUMO
Enhanced expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) and 9-O-acetylated disialoganglioside (9-OAcGD3) was observed on lymphoblasts of childhood acute lymphoblastic leukemia (ALL). Sialate-O-acetyltransferase (SOAT) and sialate-O-acetylesterase (SIAE) are the two main enzymes responsible for the quantity of the O-acetyl ester groups on sialic acids (Sias). We have earlier shown an enhanced level of SOAT activity, capable of transferring acetyl groups to Sias of glycoconjugates in the microsomes of lymphoblasts of these children. We further observed a decreased SIAE activity in both lysosomal and cytosolic fractions of ALL cell lines and primary cells from bone marrow of patients compared with peripheral blood mononuclear cells from healthy donors, which preferentially hydrolyze O-acetyl groups at C-9 of Sia. The level of O-acetylated Sias in the cytosolic and the lysosomal fractions showed a good correlation with SIAE activity in the corresponding fractions. The apparent K(M) values for SIAE in the lysosomal and the cytosolic fractions from lymphoblasts of ALL patients are 0.38 and 0.39 mM, respectively. These studies demonstrate that both SIAE and SOAT activities seem to be responsible for the enhanced level of Neu5,9Ac(2) in lymphoblasts, which is a hallmark in ALL. This was subsequently confirmed by using an enzyme-linked immunosorbent assay that also demonstrated a steady decline in SOAT activities even in cell lysates of lymphoblasts during successful chemotherapy, like radioactive methods have shown earlier.
Assuntos
Acetilesterase/metabolismo , Aciltransferases/metabolismo , Linfócitos/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Ácidos Siálicos/metabolismo , Acetilação , Acetilesterase/química , Acetilesterase/genética , Aciltransferases/química , Adolescente , Estudos de Casos e Controles , Linhagem Celular , Criança , Pré-Escolar , Citosol/enzimologia , Feminino , Gangliosídeos/metabolismo , Expressão Gênica , Humanos , Hidrólise , Lactente , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Leucócitos Mononucleares/enzimologia , Linfócitos/metabolismo , Lisossomos/enzimologia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Ácidos Siálicos/química , Sialoglicoproteínas/metabolismoRESUMO
Childhood acute lymphoblastic leukaemia is characterized by aberrant proliferation and accumulation of malignant lymphoblasts in bone marrow (BM), followed by their migration into circulation. An enhanced cell-surface expression of ALL-associated 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) was demonstrated. Present investigation reports a positive correlation between the increased density of Neu5,9Ac(2)-GPs on lymphoblasts and their mobilization from BM involving enhanced Neu5,9Ac(2) on CD45 demonstrating modulation of FAK and ERK molecules. In contrast, a small population of cells, identified as haematopoietic precursors, with comparatively lesser Neu5,9Ac(2)-GPs showed increased binding towards BM stroma. Thus, Neu5,9Ac(2)-GPs is a developmentally regulated oncofoetal antigen, whose up-regulation is imperative in the interaction between lymphoblasts and BM stroma, governing their mobilization into circulation.
Assuntos
Medula Óssea/metabolismo , Movimento Celular , Proliferação de Células , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Sialoglicoproteínas/metabolismo , Acetilação , Western Blotting , Medula Óssea/patologia , Adesão Celular , Criança , Humanos , Antígenos Comuns de Leucócito/metabolismo , Linfócitos/patologia , Prognóstico , Ácidos Siálicos/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Regulação para CimaAssuntos
Células Eritroides/metabolismo , Gangliosídeos/metabolismo , Linfócitos/metabolismo , Acetilação , Animais , Anticorpos Antifosfolipídeos/metabolismo , Apoptose/efeitos dos fármacos , Sequência de Carboidratos , Caspase 3/metabolismo , Membrana Celular/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Eritroides/citologia , Eritropoese/fisiologia , Gangliosídeos/química , Gangliosídeos/farmacologia , Humanos , Linfócitos/citologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Fragilidade Osmótica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Ceramide is an important second messenger that has diverse cellular and biological effect. It is a specific and potent inducer of apoptosis and suppressor of cell growth. In leukemia, chemoresistance generally developed due to deregulated ceramide metabolism. In combinatorial treatment strategies of leukemia, few components have the capability to increases ceramide production. Manipulation in ceramide production by physiological and pharmacological modulators therefore will give additive effect in leukemia chemotherapy. RESULTS: Here, we show that Withanolide D (C4ß-C5ß,C6ß-epoxy-1-oxo-,20ß, dihydroxy-20S,22R-witha-2,24-dienolide; WithaD), a pure herbal compound isolated from Withania somnifera could effectively induces apoptosis in a dose and time dependant manner both in myeloid (K562) and lymphoid (MOLT-4) cells being nontoxic to normal lymphocytes and control proliferative cells. WithaD potentially augment ceramide production in these cells. Downstream of ceramide, WithaD acted on MKK group of proteins and significantly increased JNK and p38MAPK phosphorylation. Pharmacological inhibition of p38MAPK and JNK proves their cooperative action on WithaD-induced cell death. Dissecting the cause of ceramide production, we found activation of neutral sphingomyelinase and showed neutral-sphingomyelinase 2 (N-SMase 2) is a critical mediator of WithaD-induced apoptosis. Knockdown of N-SMase 2 by siRNA and inhibitor of N-SMase (GW4869) significantly reduced WithaD-induced ceramide generation and phosphorylation of MKK4 and MKK3/6, whereas phosphorylation of MKK7 was moderately regulated in leukemic cells. Also, both by silencing of N-SMase 2 and/or blocking by GW4869 protects these cells from WithaD-mediated death and suppressed apoptosis, whereas Fumonisin B1, an inhibitor of ceramide synthase, did not have any effect. Additionally, WithaD effectively induced apoptosis in freshly isolated lymphoblasts from patients and the potent cell killing activity was through JNK and p38MAPK activation. CONCLUSION: Our results demonstrate that WithaD enhance the ceramide accumulation by activating N-SMase 2, modulate phosphorylation of the JNK and p38MAPK and induced apoptosis in both myeloid and lymphoid cells along with primary cells derived from leukemia patients. Taken together, this pure herbal compound (WithaD) may consider as a potential alternative tool with additive effects in conjunction with traditional chemotherapeutic treatment, thereby accelerate the process of conventional drug development.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Vitanolídeos/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Compostos de Anilina/farmacologia , Compostos de Benzilideno/farmacologia , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Leucemia/tratamento farmacológico , Leucemia/genética , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 6/metabolismo , MAP Quinase Quinase 7/metabolismo , Microscopia Eletrônica de Varredura , Fosforilação/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34(+) cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Carbazóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Receptor Cross-Talk/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor fas/fisiologia , Alcaloides/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Carbazóis/química , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Recém-Nascido , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/fisiologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta , Receptor Cross-Talk/fisiologia , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Membrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (-30 to 40%) and enzymatic activity toward ganglioside GD1a (-25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (-55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0-G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9-OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse.
Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Proteínas de Membrana/metabolismo , Neuraminidase/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Adolescente , Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Criança , Pré-Escolar , Progressão da Doença , Regulação para Baixo , Feminino , Citometria de Fluxo , Fluorometria , Humanos , Lactente , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/enzimologia , Masculino , Proteínas de Membrana/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismoRESUMO
Altered sialylation occurs in essentially all types of human and experimental cancers. Although, aberrant sialylation is believed to mainly due to altered sialyltransferase (ST) level, so far, expression pattern of different STs in acute lymphoblastic leukemia has never been investigated. Accordingly, the aim of our study was to monitor the changes in mRNA expression of ST6Gal I, ST3Gal V and ST8Sia I in patients by real-time PCR, which may provide prognostic information useful in defining appropriate therapeutic options. Our data demonstrated that ST6Gal I and ST3Gal V mRNA were up-regulated in lymphoblasts whereas its presence was negligible in non-malignant donors. In contrast, ST8SiaI was downregulated in patients. The extents of linkage-specific sialylation of glycoconjugates were found to be associated with disease establishment. Additionally, ST6Gal I and ST3Gal V were positively correlated with the high risk of the disease (P=0.0032 and 0.0016). This differential ST level can be used as biomarker with the molecular method of quantitative PCR and may be useful to discriminate normal and cancer patients.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Sialiltransferases/genética , Adolescente , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Lactente , Células Jurkat , Masculino , Prognóstico , RNA Mensageiro/análise , Fatores de Risco , Células Tumorais Cultivadas , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and ß-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEß-thalassemia.
Assuntos
Apoptose , Eritrócitos/patologia , Glicoconjugados/sangue , Esferocitose Hereditária/sangue , Talassemia alfa/sangue , Talassemia beta/sangue , Acetilglucosamina/sangue , Envelhecimento Eritrocítico , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , Fosfatidilserinas/sangue , Ácidos Siálicos/sangueRESUMO
Point mutations of alpha-globin genes in homozygous or in compound heterozygous states cause severe alpha-thalassemia (alpha-thal). Here we describe a polymerase chain reaction-restriction fragment length polymorphism-based method for easy detection of the point mutation Hb Sallanches [alpha104(G11)Cys-->Tyr, TGC>TAC], earlier detected by a sequencing technique. In a cohort of 104 unrelated putative alpha-thal patients, nine carried the mutation and two were homozygotes. The mutation occurred on both the alpha2- or alpha1-globin genes. The phenotypes, in conjunction with other point mutations or deletions, are presented. Earlier detected in Pakistan and Punjab of India, it is probably present all over the Indian subcontinent.
Assuntos
Hemoglobinas Anormais/genética , Talassemia alfa/genética , Frequência do Gene , Humanos , Incidência , Índia/epidemiologia , Fenótipo , Mutação Puntual , Polimorfismo de Fragmento de RestriçãoRESUMO
Enhanced levels of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)GPs) as disease-associated molecules was reported to act as signaling molecules for promoting survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Here, we searched for potential physiological ligands for Neu5,9Ac(2)GPs that could be involved in modulating the survival of lymphoblasts. Accordingly, we examined the presence of binding proteins for Neu5,9Ac(2)GPs on cell lines and primary cells of patients with B- and T-ALL, at presentation of the disease. Peripheral blood mononuclear cells from normal healthy donors and cells from myeloid leukemia patients were used for comparison. Neu5,9Ac(2)GPs-binding proteins (BPs) were specifically detected on the surface of both T- and B-ALL-lymphoblasts and ALL-cell lines along with the consistent presence of Neu5,9Ac(2)GPs. The Neu5,9Ac(2)GPs and BPs also co-localized on the cell surface and interacted specifically in vitro. Apoptosis of lymphoblasts, induced by serum starvation, was reversed in the presence of purified Neu5,9Ac(2)GPs due to possible engagement of BPs, and the anti-apoptotic role of this interaction was established. This is the first report of the presence of potential physiological ligands for disease-associated molecules like Neu5,9Ac(2)GPs, the interaction of which is able to trigger an anti-apoptotic signal conferring a survival advantage to leukemic cells in childhood ALL.
Assuntos
Apoptose , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Sialoglicoproteínas/metabolismo , Acetilação , Adolescente , Adulto , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Lactente , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Ligação Proteica , Sialoglicoproteínas/farmacologia , Adulto JovemRESUMO
Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac(2)-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V (max) of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.
Assuntos
Acetiltransferases/metabolismo , Medula Óssea/enzimologia , Microssomos/enzimologia , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Acetilcoenzima A/metabolismo , Adolescente , Medula Óssea/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Monofosfato de Citidina/metabolismo , Humanos , Lactente , Masculino , Microssomos/patologia , Ácido N-Acetilneuramínico/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologiaRESUMO
Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.