RESUMEN
Several studies reported the benefits of switching from anticalcineurins to mTOR inhibitors to avoid cancer occurrence after organ transplantation. The purpose of our study was to determine in vivo biological markers to explain these benefits. Cellular changes related to cellular senescence and DNA damage were analyzed in peripheral blood lymphocytes. Thirty-five kidney transplanted patients receiving anticalcineurins were investigated: 17 patients were proposed to switch to rapamycin and 18 patients with similar age and transplantation duration, continued anticalcineurins. Rapamycin effects were studied one year after the switch. Thirteen healthy volunteers and 18 hemodialyzed patients were evaluated as control. Compared with the healthy group, hemodialyzed and transplanted patients exhibited a significant decrease in telomere length, an increase in p16(INK4A) mRNA expression and in lymphocytes with 53BP1 foci. A destabilization of the shelterin complexes was suggested by a significant TIN2 mRNA decrease in transplanted patients compared with controls and a significant increase in TRF1, TRF2 and POT1 expression in switch-proposed patients compared with the non-switched subgroup. Rapamycin treatment resulted in a significant decrease in DNA damage and a slight TIN2 increase. In vitro experiments strengthened in vivo results showing that rapamycin but not FK506 induced a significant DNA damage decrease and TIN2 expression increase compared with controls. The roles of rapamycin in the decrease in DNA damage in vivo and the rescue of shelterin gene expression are demonstrated for the first time. These data provide new insights into understanding of how rapamycin may overcome genomic injuries.
Asunto(s)
Daño del ADN/efectos de los fármacos , Trasplante de Riñón/métodos , Linfocitos/efectos de los fármacos , Sirolimus/uso terapéutico , Anciano , Inhibidores de la Calcineurina/efectos adversos , Inhibidores de la Calcineurina/uso terapéutico , Femenino , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , Enfermedades Renales/sangre , Enfermedades Renales/cirugía , Trasplante de Riñón/efectos adversos , Linfocitos/ultraestructura , Masculino , Persona de Mediana Edad , Telómero/efectos de los fármacosRESUMEN
New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment.
Asunto(s)
Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dexametasona/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Anciano , Anciano de 80 o más Años , Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Piperidinas , Estrés Fisiológico/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Adult Acute Lymphoblastic Leukemia (ALL) therapies have been improved by pediatric-like approaches. However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The p16(INK4A)/CDK4-6/pRb pathway and telomerase activity, which are implicated in cell activation and aging, were analyzed to identify new prognostic markers. Proteins of the p16(INK4A)/CDK4-6/pRb pathway and telomerase activity were analyzed in 123 adult B-cell precursor (BCP) ALL cases included in the GRAALL/GRAAPH trials. We found a significantly increased expression of p16(INK4A) in BCP-ALLs with MLL rearrangement. Telomerase activity was significantly lower in Philadelphia chromosome-negative/IKAROS-deleted (BCR-ABL1(-)/IKAROS(del)) cases compared to Philadelphia chromosome-positive (BCR-ABL1+) BCP-ALLs. In BCR-ABL1+ ALLs, high CDK4 expression, phosphorylated pRb (p-pRb) and telomerase activity were significantly associated with a shorter disease-free survival (DFS) and event-free survival (EFS). Enhanced p16(INK4A) expression was only related to a significantly shorter DFS. In vitro analyses of normal stimulated lymphocytes after short- and long-term cultures demonstrated that the observed protein variations of poor prognosis in BCR-ABL1+ ALLs may be related to cell activation but not to cell aging. For these patients, our findings argue for the development of therapeutic strategies including the addition of new lymphocyte activation inhibitors to current treatments.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína de Retinoblastoma/metabolismo , Telomerasa/metabolismo , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Análisis Citogenético , Femenino , Estudios de Seguimiento , Humanos , Inmunofenotipificación , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Adulto JovenRESUMEN
The p16(INK4a) protein is considered to regulate the cell cycle progression mainly by inhibiting cyclin-dependent kinases (CDKs) 4 and 6 activity and leading to an arrest in G(0)/G(1). Here, we report that ectopic expression of p16(INK4a) in three p16-/pRb(Wt)/p53(Wt) human cancer cell lines MCF7, U2OS and U87 induces S-phase lengthening along with G(1) accumulation. S-phase lengthening is suggested by the discrepancy between the unchanged or even increased percentage of cells in S phase found by flow cytometry DNA content analysis and the drop of BrdU labelling, and demonstrated by IdU/BrdU double labelling. p16(INK4a) induces a profound decrease in the CDK4/6-mediated pRb phosphorylation on Ser-807/811, a downregulation of CDK2 and CDK1 protein expression independently of G(1) accumulation, and a decrease in Thr/Pro phosphorylation in part carried out by CDKs. In MCF7 cells, overexpression of the p16 G101W mutant, which is unable to inhibit CDK4/6 kinase activity and shows a modified subcellular localization, does not provoke the S-phase lengthening and the inhibition of Ser807/811-pRB and of Thr/Pro phosphorylation as wild-type p16(INK4a) does. Our results demonstrate that p16(INK4a) induces a S-phase lengthening independently of cellular origin. The CDK4/6 kinase activity inhibition together with the reduced expression of CDK2 and CDK1 acting downstream of G(1) phase may prevent cells from any possible kinasic compensatory mechanisms, and thus lead to a cell cycle progression inhibition.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase S , Proteína p53 Supresora de Tumor/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Fase G1 , Humanos , Mutagénesis Sitio-Dirigida , FosforilaciónRESUMEN
Indirubin-3'-monoxime (IO) is a derivative of indirubin, an active compound of a traditional Chinese medicinal recipe used to treat various inflammatory and malignant diseases. The main in vitro targets of IO (i.e. cyclin dependent kinases, glycogen synthase kinase-3beta, Stat 3 and Aryl hydrocarbon receptor) are regulators of lymphocyte activation. We investigated the interest of IO and its derivative 6-bromo-indirubin-3'oxime (6BIO) for inhibiting the growth of malignant lymphoid cells. IO (1-20 microM) induced cell cycle inhibition and cell death in malignant B- (IM9, Reh6) and T- (Jurkat, CEM-T) lymphoid cell lines depending to cell type, doses, and duration of treatment. IO and 6BIO (10 microM) treatment for 24 and 48 h were compared: 6BIO treatment resulted in a stronger cytotoxicity and more profound inhibition of cell proliferation. Taken together, these results showed that IO and, moreover, its derivative 6BIO may be potent antiproliferative agents in malignant lymphoid cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Indoles/farmacología , Linfocitos/efectos de los fármacos , Oximas/farmacología , Antibióticos Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina E/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Linfocitos/metabolismo , Linfocitos/patología , Factores de TiempoRESUMEN
Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.