Liver transplant with controlled donors after circulatory death with normothermic regional perfusion and brain dead donors: A multicenter cohort study of transfusion, one-year graft survival and mortality.

BACKGROUND: Controlled donation after circulatory death (cDCD) has expanded the donor pool for liver transplantation (LT). However, transfusion requirements and perioperative outcomes should be elucidated. The aim of this multicenter study was to assess red blood cell (RBC) transfusions, one-year graft and patient survival after LT after cDCD with normothermic regional perfusion (NRP) compared with donors after brain death (DBD). METHODS: 591 LT carried out in ten centers during 2019 were reviewed. Thromboelastometry was used to manage coagulation and blood product transfusion in all centers. Normothermic regional perfusion was the standard technique for organ recovery. RESULTS: 447 patients received DBD and 144 cDCD with NRP. Baseline MCF Extem was lower in the cDCD group There were no differences in the percentage of patients (63% vs. 61% p = 0.69), nor in the number of RBC units transfused (4.7 (0.2) vs 5.5 (0.4) in DBD vs cDCD, p = 0.11. Twenty-six patients (6%) died during admission for LT in the DBD group compared with 3 patients (2%) in the cDCD group (p = 0.15). To overcome the bias due to a worse coagulation profile in cDCD recipients, matched samples were compared. No differences in baseline laboratory data, or in intraoperative use of RBC or one-year outcome data were observed between DBD and cDCD recipients. CONCLUSIONS: cDCD with NRP is not associated with increased RBC transfusion. No differences in graft and patient survival between cDCD and DBD were found. Donors after controlled circulatory death with NRP can increasingly be utilized with safety, improving the imbalance between organ donors and the ever-growing demand.

Oncological Evaluation by Positron-emission Tomography, Circulating Tumor Cells and Alpha Fetoprotein in Patients With Hepatocellular Carcinoma on the Waiting List for Liver Transplantation.

INTRODUCTION: The objectives of this study are the determination of the number of circulating tumor cells (CTCs), by means of the IsoFlux enrichment system (Fluxion Biosciences Inc, San Francisco, California, United States) in patients with hepatocellular carcinoma (HCC) in compliance with the Milan criteria and on the waiting list for hepatic transplantation, as well as the study of its relation with the of α-fetoprotein levels (AFP) and positron-emission tomography-computed tomography (PET-CT) findings. PATIENTS AND METHODS: An oncologycal evaluation with PET-CT, CTCs, and AFP was conducted in 24 consecutive patients with HCC eligible for orthotopic liver transplantation according to the Milan criteria. The diagnosis of HCC was made according to clinical, biological, and radiological findings. RESULTS: We detected CTCs in peripheral blood in 21 of 24 patients (87.5%) before liver transplantation, with a mean number CTCs of 156 ± 370 (range, 2 to 1768) with statistically significant association between number of CTCs detected in peripheral blood and the time within the waiting list (P < .05), but not betwen AFP levels and standard uptake value and time to orthotopic liver transplantation (P > .05). CONCLUSIONS: PET-TC, CTCs, and AFP levels could be an essential key for the correct management of the patients with HCC on the waiting list for liver transplantation.

Inflammasome-dependent IL-1ß release depends upon membrane permeabilisation.

Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

Comparison of Two Types of Liquid Biopsies in Patients With Hepatocellular Carcinoma Awaiting Orthotopic Liver Transplantation.

INTRODUCTION: Orthotopic liver transplantation (OLT) is considered one of the few curative treatments available for early stages of hepatocellular carcinoma (HCC). It has been shown that more than 10% of transplanted individuals suffer relapse during the first year after surgery and most of them die because of the tumor. The circulating tumor cells (CTCs) are the main source of recurrences as they disseminate from a primary or metastatic tumor lesion through peripheral blood. We aimed to determine the concentration of CTCs in peripheral blood in these patients by 2 different approaches: the CellSearch and the IsoFlux systems to assess their applicability to this disease monitoring. PATIENTS AND METHODS: A comparative study was conducted in 21 patients with HCC eligible for liver transplantation according to the Milan criteria, whose peripheral blood was processed by the CellSearch and the IsoFlux systems. RESULTS: CTCs were isolated in 1 of the 21 patients (4.7%) by the CellSearch system and in 19 of the 21 patients (90.5%) by the IsoFlux method. The comparison of both methods using Bland-Altman plot shows that there is not consistency in the determination of CTCs in our patients, finding a proportional bias between them. CONCLUSION: The results obtained by both CTCs isolation systems are not interchangeable nor transferable. The CellSearch system does not seem to be the ideal approach for studying CTCs in patients with HCC. The IsoFlux system displays greater sensitivity in the identification of CTCs and might become an important tool in patient management.

[Effectiveness of plerixafor in patients undergoing mobilization autologous haematopoietic progenitor cell]. / Efectividad de plerixafor en pacientes sometidos a movilización de progenitores hematopoyéticos para autotrasplante.

OBJECTIVE: To evaluate effectiveness of treatment with plerixafor in patients undergoing posterior mobilization for hematopoietic transplant at our hospital. METHODS: Retrospective study of all patients who until September 2012, received plerixafor in their scheme of mobilization into peripheral blood hematopoietic progenitors. We reviewed the medical records and records of drug dispensing outpatient consultation. Effectiveness variables used were: CD34/kg in apheresis product obtained, and day dose received colony stimulating factor (G-CSF) and Plerixafor. Each patient was compared to the effectiveness of the drug results with those obtained earlier mobilization schemes where Plerixafor not used, if present. Data were analyzed using IBM SPSS v19. RESULTS: A total of 24 patients received plerixafor in our hospital. Diagnoses were distributed: 15 NHL, 6 patients with multiple myeloma, 2 Hodgkin's disease, and one metastatic choriocarcinoma. The effectiveness outcomes were no plerixafor mobilization (n = 18): 5 patients were mobilized with G-CSGF only, 13 with G-CSF and chemotherapy. The G-CSF dose / day was mcg 931.1 (± 179.5) for 9.5 days (± 4.7). The average product obtained CD34/kg in cells was 0.2 (± 0.5). No patient received sufficient product (≥ 2 x 106 cells/kg) for subsequent autologous transplantation. 100% of the demonstrations failed. Mobilization with plerixafor (n = 24): 13 patients were mobilized with GCSGF only, 11 with G-CSF and chemotherapy. The G-CSF dose/ day and averaged Plerixafor mcg 885.1 (± 240.1) and 19.8 (± 4.4), respectively, administered for 8.9 (± 5.1) and 1 , 5 (± 0.6) days, respectively. The average product obtained in CD34/kg was 2.3 x 106 cells (± 1.7) (p = 0.014 in relation to the demonstrations without Plerixafor). Only 12.5% (n = 3) patients were unable to undergo autologous transplant. CONCLUSIONS: In our patients, plerixafor has proven effective in mobilizing hematopoietic progenitors for autologous back.

Objetivo: Evaluar efectividad del tratamiento con plerixafor en pacientes sometidos a movilización para posterior autotrasplante de progenitores hematopoyéticos en nuestro hospital. Métodos: Estudio retrospectivo de todos los pacientes que hasta septiembre 2012, recibieron plerixafor en su esquema de movilización de progenitores hematopoyéticos a sangre periférica. Se revisaron las historias clínicas y los registros de dispensación de medicamentos de la consulta de pacientes externos. Las variables de efectividad utilizadas fueron: CD34/kg en producto de aféresis obtenidas, dosis y días recibidos de factor estimulante de colonias (G-CSF) y de plerixafor. Para cada paciente se comparó los resultados de efectividad del fármaco con los obtenidos para anteriores esquemas de movilización en los que no se utilizó plerixafor, en caso de tenerlos. Los datos se analizaron mediante IBM spss v19. Resultados: Un total de 24 pacientes recibieron plerixafor en nuestro hospital. Los diagnósticos se distribuyeron: 15 linfoma no Hodgkin , 6 pacientes con mieloma múltiple, 2 enfermedad de Hodgkin, y 1 coriocarcinoma diseminado. Los resultados de efectividad fueron: Movilización sin plerixafor (n = 18): 5 pacientes se movilizaron sólo con G-CSGF, 13 con G-CSF y quimioterapia. La dosis de G-CSF /día fue de 931,1 mcg (± 179,5), durante 9,5 días (± 4,7). El promedio de CD34/kg en producto obtenido fue de 0,2 células (± 0,5). Ningún paciente obtuvo producto suficiente (≥?2 x 106 células/kg) para el posterior autotrasplante. El 100 % de las movilizaciones fracasaron. Movilización con plerixafor (n = 24): 13 pacientes se movilizaron sólo con G-CSGF, 11 con G-CSF y quimioterapia. La dosis de G-CSF /día y de plerixafor promedio fue de 885,1 mcg (± 240,1) y 19,8 (± 4,4), respectivamente, administrados durante 8,9 (± 5,1) y 1,5 (± 0,6) días, respectivamente. El promedio de CD34/kg en producto obtenido fue de 2,3x106 células (±1,7) (p = 0,014, en relación a las movilizaciones sin plerixafor). Sólo el 12,5% (n = 3) pacientes no pudieron someterse a autotrasplante. Conclusiones: En nuestros pacientes, plerixafor ha demostrado ser efectivo en la movilización de progenitores hematopoyéticos para posterior autotrasplante.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

Coroidopatía serosa central crónica. Tratamiento combinado con Ranibizumab y terapia fotodinámica / Chronic central serous chorioretinopathy. Combined treatment with Ranibizumab and photodynamic therapy

Caso Clínico. Varón de 72 años que acude a consulta refiriendo visión borrosa en ojo derecho (OD) de más de dos años de evolución y que presentaba un desprendimiento neurosensorial (DNS) macular en la tomografía de coherencia óptica (OCT). Habiendo sido diagnosticado previamente de coroidopatía serosa central (CSC) crónica en otro centro y habiendo sido tratado con ranibizumab intravítreo y fotocoagulación láser. Decidimos combinar el tratamiento con ranibizumab intravítreo y terapia fotodinámica para evitar nuevas reactivaciones. Discusión. Tras dos sesiones de terapia fotodinámica junto con tres dosis de Ranibizumab el cuadro se controló estabilizándose la agudeza visual del paciente. Ambas estrategias combinadas dieron buen resultado, disminuyendo el número de brotes en los últimos meses; no obstante debemos continuar con el seguimiento para observar posibles efectos adversos a medio o largo plazo (AU)

Case Report. 72 years-old male who came to our service because of blurred vision in his right eye (OD), and who presented a neurosensorial detachment (NSD) in optical coherence tomography (OCT). Having already been diagnosed in other center of chronic central serous chorioretinopathy and having already been treated with intravitreous Ranibizumab and photocoagulation laser. We decided to combine intravitreous Ranibizumab treatment and photodynamic therapy in order to avoid new reactivations. Discussion. After two photodynamic therapy sessions and three intravitreous Ranibizumab inyections the patient´s visual acuity got stable. We got good results combining both therapeutical strategies, and the number of outbreaks has decreased during the last months; however we should carry on checking our patient to detect any possible half or long term side effects (AU)

What do we know about the clinical impact of complete withdrawal of immunosuppression in liver transplantation?

The liver is a privileged organ with a lower incidence of rejection than other organs. However, immunosuppressive regimens are still required to control the alloreactive T-lymphocyte response after transplantation. These treatments may lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases, and chronic renal insufficiency. In clinical transplantation, there is increasing evidence that some liver transplant recipients who cease taking immunosuppressive drugs maintain allograft function, suggesting that tolerance is already present. This strategy is feasible in 25% to 33% of liver transplant recipients. Few of the studies performed so far have provided a detailed analysis of the impact of immunosuppression (IS) withdrawal on pre-existing complications derived from the long-term administration of immunosuppressive drugs and the side effects associated with it. In preliminary studies, IS withdrawal was safely achieved in selected liver transplant patients, and improved not only kidney function, but also other IS-associated side-effects such as hypercholesterolemia, hyperuricemia, hypertension, and diabetes control. However, longer follow-up periods are needed to confirm the benefits of IS withdrawal in liver transplant patients.

Dual effects of ß3 integrin subunit expression on human pancreatic cancer models.

BACKGROUND: Pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration. METHODS: In view of these findings, we examined the role of ß(3) in pancreatic cancer by generating two stable ß(3)-expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo. RESULTS: Transduction of ß(3) selectively augmented the functional membrane α(v)ß(3) integrin levels, as evident from the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected in ß(3)-overexpressing cells. Moreover, tumors expressing ß(3) displayed reduced growth. Interestingly, treatment of mice with an α(v)-blocking antibody inhibited the growth of ß(3)-expressing tumors to a higher extent. CONCLUSIONS: Our results collectively support the hypothesis that α(v)ß(3) integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors.

p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors.

p16(Ink4a) is a protein involved in regulation of the cell cycle. Currently, p16(Ink4a) is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16(Ink4a) has also been described in several tumors. This review attempts to elucidate when and why p16(Ink4a) overexpression occurs, and to suggest possible implications of p16(Ink4a) in the diagnosis, prognosis and treatment of cancer.

P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness.

ATP-gated P2X(7) receptors (P2X(7)R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X(7)R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X(7)R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X(7) receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca(2+)-activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X(7)R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X(7)R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X(7)R antagonists.

Impact of a CAD system in a screen-film mammography screening program: a prospective study.

OBJECTIVE: The purpose of our study was to perform a prospective assessment of the impact of a CAD system in a screen-film mammography screening program during a period of 3 years. MATERIALS AND METHODS: Our study was carried out on a population of 21,855 asymptomatic women (45-65 years). Mammograms were processed in a CAD system and independently interpreted by one of six radiologists. We analyzed the following parameters: sensitivity of radiologist's interpretation (without and with CAD), detection increase, recall rate and positive predictive value of biopsy, CAD's marks, radiologist's false negatives and comparative analysis of carcinomas detected and non-detected by CAD. RESULTS: Detection rate was 4.3‰. CAD supposed an increase of 0.1‰ in detection rate and 1% in the total number of cases (p<0.005). The impact on recall rate was not significant (0.4%) and PPV of percutaneous biopsy was unchanged by CAD (20.23%). CAD's marks were 2.7 per case and 0.7 per view. Radiologist's false negatives were 13 lesions which were initially considered as CAD's false positives. CONCLUSIONS: CAD supposed a significant increase in detection, without modifications in recall rates and PPV of biopsy. However, better results could have been achieved if radiologists had considered actionable those cases marked by CAD but initially misinterpreted.

Dual effects of ß3 integrin subunit expression on human pancreatic cancer models.

BACKGROUND: pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration. METHODS: in view of these findings, we examined the role of ß3 in pancreatic cancer by generating two stable ß3-expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo. RESULTS: transduction of ß3 selectively augmented the functional membrane αvß3 integrin levels, as evident from the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected in ß3-overexpressing cells. Moreover, tumors expressing ß3 displayed reduced growth. Interestingly, treatment of mice with an αv-blocking antibody inhibited the growth of ß3-expressing tumors to a higher extent. CONCLUSIONS: our results collectively support the hypothesis that αvß3 integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors.

Loss of poly(ADP-ribose) polymerase-2 leads to rapid development of spontaneous T-cell lymphomas in p53-deficient mice.

Poly(ADP-ribose) polymerase-2 (Parp-2) belongs to a family of enzymes that catalyse poly(ADP-ribosyl)ation of proteins. Parp-2 deficiency in mice (Parp-2(-/-)) results in reduced thymic cellularity associated with increased apoptosis in thymocytes, defining Parp-2 as an important mediator of T-cell survival during thymopoiesis. To determine whether there is a link between Parp-2 and the p53 DNA-damage-dependent apoptotic response, we have generated Parp-2/p53-double-null mutant mice. We found that p53(-/-) backgrounds completely restored the survival and development of Parp-2(-/-) thymocytes. However, Parp-2-deficient thymocytes accumulated high levels of DNA double-strand breaks (DSB), independently of the p53 status, in line with a function of Parp-2 as a caretaker promoting genomic stability during thymocytes development. Although Parp-2(-/-) mice do not have spontaneous tumours, Parp-2 deficiency accelerated spontaneous tumour development in p53-null mice, mainly T-cell lymphomas. These data suggest a synergistic interaction between Parp-2 and p53 in tumour suppression through the role of Parp-2 in DNA-damage response and genome integrity surveillance, and point to the potential importance of examining human tumours for the status of both genes.

Análisis de los resultados clínicos y la carga asistencial del screening oportunista en una Unidad de Diagnóstico por la Imagen de la Mama / Analysis of the clinical results and the nursing workloads and costs of opportunist screening in a Breast Diagnostic Imaging Unit

El objetivo de este trabajo es analizar el impacto diagnóstico y la carga asistencial del screening oportunista (SO). Se analizó la actividad desarrollada entre 2003 y 2006. Se estudiaron 24.317 pacientes (9.487 remitidas como SO) y se realizaron44.269 mamografías en el Programa Poblacional. El SO supuso el 42% de la demanda, 39% de pacientes, 33,69% de estudios y 22,32% de costes. El coste/carcinoma fue 964 URVs, frente a 461,16 URVs en Programa y 195,64 URVs en sintomáticas. El SO supone un alto volumen de actividad y consumo de recursos. Es necesario reducir la carga asistencial promoviendo la participación en Programas e introduciendo estrategias de gestión para reducir costes (AU)

The objective of this work is to analyse the diagnostic impact and nursing workloads and costs of opportunist screening (OS). After analyzing the diagnostic impact and nursing workloads and costs of opportunist screening (OS). After analyzing the activity carried out between 2003 and 2006, a high volume of activity and consumption of resources is observed. It thus concludes that it is necessary to reduce the nursing workloads and costs encouraging the participation in Programmes and introducing management strategies to reduce costs (AU)

Expression profiles of a human pancreatic cancer cell line upon induction of apoptosis search for modulators in cancer therapy.

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.

The fate of pancreatic tumor cell lines following p16 overexpression depends on the modulation of CDK2 activity.

Restitution of lost tumor-suppressor activities may be a promising strategy to target specifically cancer cells. However, the action of ectopically expressed tumor-suppressor genes depends on genetic background of tumoral cells. Ectopic expression of p16(INK4a) induces either cell cycle arrest or apoptosis in different pancreatic cancer cell lines. We examined the molecular mechanisms mediating these two different cellular responses to p16 overexpression. Ectopic expression of p16 leads to G1 arrest in NP-9 cells by redistributing p21/p27 CKIs and inhibiting cyclin-dependent kinase CDK2 activity. In contrast, in NP-18 cells cyclin E (CycE)/CDK2 activity is significantly higher and is not downregulated by p16-mediated redistribution of p21/p27. Moreover, inhibition of CDK4 activity with fascaplysine, which does not affect CycE/CDK2 activity, reduces pocket protein phosphorylation in both cell lines, but fails to induce growth arrest. Like overexpression of p16, fascaplysine induces apoptosis in NP-18 cells, suggesting that inhibition of D-type cyclin/CDK activity in cells with high levels of CycE/CDK2 activity activates an apoptotic pathway. Inhibition of CycE/CDK2 activity via ectopic expression of p21 in NP-18 cells overexpressing p16 induces growth arrest and prevents p16-mediated apoptosis. Accordingly, silencing of p21 expression by using small interfering RNA switches the fate of p16-expressing NP-9 cells from cell cycle arrest to apoptosis. Our data suggest that, after CDK4/6 inactivation, the fate of pancreatic tumor cells depends on the ability to modulate CDK2 activity.

ALL-1/MLL1, a homologue of Drosophila TRITHORAX, modifies chromatin and is directly involved in infant acute leukaemia.

Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of >50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX.

Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements.

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16(INK4a/CDKN2), a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt-p16 cDNA (Ad5RSV-p16). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment.