Neutron spectroscopy of plutonium using a handheld detection system.

The ability to distinguish multiple forms of plutonium from one another, such as oxide and metal, is paramount in areas of nuclear nonproliferation and international safeguards. In its metal form, plutonium can be readily used in a nuclear weapon, while oxide forms are associated with nuclear reactor fuel. Oxide-based plutonium forms emit neutrons with an energy spectrum that is significantly different from the fission neutrons that are emitted from plutonium metal. Organic scintillation detectors output pulses that are proportional to the neutron energy deposited, and therefore present a means of distinguishing these plutonium forms based on their energy spectra. In this work, metal and oxide forms of plutonium were measured using a handheld detection system based on an organic glass scintillator. Monte Carlo modeling of these experiments was performed to provide insight into the origin of the features in the observed light output spectra. Through analysis of multiple regions of these spectra, in a matter of minutes we were able to unambiguously discriminate oxide and metal plutonium forms from one another and from a plutonium-beryllium neutron source, which was considered for comparison because these sources are commonly used in industrial applications. The ability to discriminate weapons-usable material from nuclear reactor fuel has applications in nuclear treaty verification and safeguards.

Angular distribution of neutron production by proton and carbon-ion therapeutic beams.

Carbon-ion beams are increasingly used in the clinical practice for external radiotherapy treatments of deep-seated tumors. At therapeutic energies, carbon ions yield significant secondary products, including neutrons, which may be of concern for the radiation protection of the patient and personnel. We simulated the neutron yield produced by proton and carbon-ion pencil beams impinging on a clinical phantom at three different angles: 15°, 45° and 90°, with respect to the beam axis. We validated the simulated results using the measured response of organic scintillation detectors. We compared the results obtained with FLUKA 2011.2 and MCNPX 2.7.0 based on three different physics models: Bertini, Isabel, and CEM. Over the different ions, energies, and angles, the FLUKA simulation results agree better with the measured data, compared to the MCNPX results. Simulations of carbon ions at low angles exhibit both the highest deviation from measured data and inter-model discrepancy, which is probably due to the different treatment of the pre-equilibrium stage. The reported neutron yield results could help in the comparison of carbon-ion and proton treatments in terms of secondary neutron production for radiation protection applications.

THE NATIONAL RADON ARCHIVE AS A USEFUL TOOL FOR DEVELOPING AND UPDATING THE NATIONAL RADON ACTION PLAN.

International recommendations and regulations require developing of National Radon Action Plans (NRAPs) to effectively manage the protection of workers and population from radon exposure. In Italy, a NRAP was published in 2002 and several activities have been carried out in this framework. Information and data regarding these and previous activities have been collected in a National Radon Archive (NRA). Activities carried out by institutionally involved institutes and agencies include several national and regional surveys, involving more than 50 000 indoor environments (dwellings, schools and workplaces), and remedial actions performed in ~350 buildings, largely in schools. Data collected in the NRA allowed also to estimate that lung cancer deaths attributable to radon exposure in Italy are ~3400 per year. On-going developments of the Italian NRA finalized to effectively use it as tool for developing, monitoring and updating the NRAP are also described.

A scintillator-based approach to monitor secondary neutron production during proton therapy.

PURPOSE: The primary objective of this work is to measure the secondary neutron field produced by an uncollimated proton pencil beam impinging on different tissue-equivalent phantom materials using organic scintillation detectors. Additionally, the Monte Carlo code mcnpx-PoliMi was used to simulate the detector response for comparison to the measured data. Comparison of the measured and simulated data will validate this approach for monitoring secondary neutron dose during proton therapy. METHODS: Proton beams of 155- and 200-MeV were used to irradiate a variety of phantom materials and secondary particles were detected using organic liquid scintillators. These detectors are sensitive to fast neutrons and gamma rays: pulse shape discrimination was used to classify each detected pulse as either a neutron or a gamma ray. The mcnpx-PoliMi code was used to simulate the secondary neutron field produced during proton irradiation of the same tissue-equivalent phantom materials. RESULTS: An experiment was performed at the Loma Linda University Medical Center proton therapy research beam line and corresponding models were created using the mcnpx-PoliMi code. The authors' analysis showed agreement between the simulations and the measurements. The simulated detector response can be used to validate the simulations of neutron and gamma doses on a particular beam line with or without a phantom. CONCLUSIONS: The authors have demonstrated a method of monitoring the neutron component of the secondary radiation field produced by therapeutic protons. The method relies on direct detection of secondary neutrons and gamma rays using organic scintillation detectors. These detectors are sensitive over the full range of biologically relevant neutron energies above 0.5 MeV and allow effective discrimination between neutron and photon dose. Because the detector system is portable, the described system could be used in the future to evaluate secondary neutron and gamma doses on various clinical beam lines for commissioning and prospective data collection in pediatric patients treated with proton therapy.

Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update.

This is a report of 148 patients with hematologic malignancies who received an unmanipulated haploidentical bone marrow transplant (BMT), followed by post-transplant high-dose cyclophosphamide (PT-CY). All patients received a myeloablative conditioning consisting of thiotepa, busulfan, fludarabine (n=92) or TBI, fludarabine (n=56). The median age was 47 years (17-74); 47 patients were in first remission (CR1), 37 in second remission (CR2) and 64 had an active disease; all patients were first grafts. The diagnosis was acute leukemia (n=75), myelodisplastic syndrome (n=24), myelofibrosis (n=16), high-grade lymphoma (n=15) and others (n=18). GVHD prophylaxis consisted in PT-CY on days +3 and +5, cyclosporine (from day 0), and mycophenolate (from day +1). The median day for neutrophil engraftment was day +18 (13-32). The cumulative incidence of grades II-IV acute GVHD was 24%, and of grades III-IV GVHD 10%. The incidence of moderate-severe chronic GVHD was 12%. With a median follow-up for the surviving patients of 313 days (100-1162), the cumulative incidence of transplant-related mortality (TRM) is 13%, and the relapse-related death is 23%. The actuarial 22 months overall survival is 77% for CR1 patients, 49% for CR2 patients and 38% for patients grafted in relapse (P<0.001). Major causes of death were relapse (22%), GVHD (2%) and infections (6%). We confirm our initial results, suggesting that a myeloablative conditioning regimen followed by unmanipulated haploidentical BMT with PT-CY, results in a low risk of acute and chronic GVHD and encouraging rates of TRM and overall survival, also for patients with active disease at the time of transplant.

miR-205 regulates basement membrane deposition in human prostate: implications for cancer development.

The basement membrane (BM) is a layer of specialized extracellular matrix that surrounds normal prostate glands and preserves tissue integrity. Lack or discontinuity of the BM is a prerequisite for tumor cell invasion into interstitial spaces, thus favoring metastasis. Therefore, BM maintenance represents a barrier against cancer development and progression. In the study, we show that miR-205 participates in a network involving ΔNp63α, which is essential for maintenance of the BM in prostate epithelium. At the molecular level, ΔNp63α is able to enhance miR-205 transcription by binding to its promoter, whereas the microRNA can post-transcriptionally limit the amount of ΔNp63α protein, mostly by affecting ΔNp63α proteasomal degradation rather than through a canonical miRNA/target interaction. Functionally, miR-205 is able to control the deposition of laminin-332 and its receptor integrin-ß4. Hence, pathological loss of miR-205, as widely observed in prostate cancer, may favor tumorigenesis by creating discontinuities in the BM. Here we demonstrate that therapeutic replacement of miR-205 in prostate cancer (PCa) cells can restore BM deposition and 3D organization into normal-like acinar structures, thus hampering cancer progression.

A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function.

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM.

Impact on survival of the number of lymph nodes removed in patients with node-negative gastric cancer submitted to extended lymph node dissection.

PURPOSE: This study was designed to establish whether the number of lymph nodes removed has an effect on prognosis in patients with node-negative gastric cancer. PATIENTS AND METHODS: We retrospectively analysed data of 114 consecutive patients who underwent gastrectomy and extended lymph node dissection for node-negative adenocarcinoma of the stomach between 2000 and 2005. Standard survival methods and restricted cubic spline multivariable Cox regression models were applied. RESULTS: Median age was 63 years and 67 patients out of 114 (59%) were males. Median number of dissected LNs was 22 (range 2-73). Median follow-up was 76 months. Patients who had ≤15 nodes removed had significantly worse distant disease-free survival, disease-free survival and overall survival at multivariable analysis than other patients. The results did not change when pT1 and pT2-3 cancer patients were analysed separately. The risk of distant metastases decreased as the number of dissected lymph nodes increased (>15). CONCLUSIONS: More extended lymph node resection offered survival benefit even in the subgroup of patients with early stage disease. Lymphadenectomy involving more than 15 lymph nodes should be performed for the treatment of node-negative gastric cancer. SYNOPSIS: The impact on survival of the number of lymph nodes removed in patients with node-negative gastric cancer has not been established. This study suggests that more extended lymph node resection offers protection, as patients who had ≤15 nodes removed had significantly worse disease-free survival and overall survival at multivariate analysis than patients in whom >15 nodes were removed.

Mutant p53 subverts p63 control over KLF4 expression in keratinocytes.

Genetic experiments established that p63 is crucial for the development and maintenance of pluri-stratified epithelia and KLF4 for the barrier function of the skin. KLF4 is one of the factors that reprogram differentiated cells to iPS. We investigated the relationship between p63 and KLF4 using RNA interference, overexpression, chromatin immunoprecipitation and transient transfections with reporter constructs. We find that p63 directly represses KLF4 in normal keratinocytes (KCs) by binding to upstream promoter sites. Unlike p63, KLF4 levels are high in the upper layers of human skin and increase upon differentiation of KCs in vitro. In HaCaT KCs, which harbor two mutant alleles of p53, inactivation of p63 and of mutant p53 leads to KLF4 repression. p63 and p53 mutants are bound to sites in the KLF4 core promoter. Importantly, expression of the H179Y and R282Q p53 mutants in primary KCs is sufficient to activate endogenous KLF4. Finally, immunohistochemical analysis of tissue arrays confirms increased coexpression of KLF4 and mutant p53 in squamous cell carcinomas. Our data indicate that suppression of KLF4 is part of the growth-promoting strategy of p63 in the lower layers of normal epidermis, and that tumor-predisposing p53 mutations hijack p63 to a different location on the promoter, turning it into an activator of this reprogramming factor.

AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition.

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.

Allogeneic hemopoietic SCT for patients with primary myelofibrosis: a predictive transplant score based on transfusion requirement, spleen size and donor type.

A total of 46 patients with primary myelofibrosis (PMF) (median age 51 years), underwent an allogeneic hemopoietic SCT (HSCT) after a thiotepa-based reduced-intensity conditioning regimen. The median follow-up for surviving patients is 3.8 years. In multivariate analysis, independent unfavorable factors for survival were RBC transfusions >20, a spleen size >22 cm and an alternative donor-24 patients had 0-1 unfavorable predictors (low risk) and 22 patients had 2 or more negative predictors (high risk). The overall actuarial 5-year survival of the 46 patients is 45%. The actuarial survival of low-risk and high-risk patients is, respectively, 77 and 8% (P<0.0001); this is because of a higher TRM for high-risk patients (RR, 6.0, P=0.006) and a higher relapse-related death (RR, 7.69; P=0.001). In multivariate Cox analysis, the score maintained its predictive value (P=0.0003), even after correcting for donor-patient age and gender, Dupriez score, IPSS (International Prognostic Scoring System) score pre-transplant and splenectomy. In conclusion, PMF patients undergoing an allogeneic HSCT may be scored according to the spleen size, transfusion history and donor type; this scoring system may be useful to discuss transplant strategies.

Prognostic models for diffuse large B-cell lymphoma in the rituximab era: a never-ending story.

BACKGROUND: Improved treatment have modified survival outcome in patients with diffuse large B-cell lymphoma (DLBCL) and altered the importance of previously recognized prognostic markers. DESIGN AND METHODS: To evaluate International Prognostic Index (IPI) score before and after rituximab introduction and to validate the absolute lymphocyte count (ALC)/revised International Prognostic Index (R-IPI) model, we carried out a retrospective analysis on a total of 831 patients with DLBCL. RESULTS: Our results show that IPI lost its discriminating power with the introduction of rituximab. The analysis of our second set allowed us to validate the ALC/R-IPI model. The R-IPI and ALC/R-IPI could still be used for designing clinical trials, but both have difficulty recognizing a high percentage of poor prognosis patients, though it remains an important goal of a good prognostic model considering the modest impact of salvage treatments on survival. CONCLUSIONS: A new model on the basis of significant variables in the rituximab era and built on a large database of patients treated with rituximab is urgently needed. As prognostic models are changing with the efficacy and mechanisms of action of treatment utilized, looking for a new prognostic score is a never-ending story in which researchers are trying to hit a continuously moving target.

Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma.

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.

Best choice of central venous insertion site for the prevention of catheter-related complications in adult patients who need cancer therapy: a randomized trial.

BACKGROUND: Central venous access is extensively used in oncology, though practical information from randomized trials on the most convenient insertion modality and site is unavailable. METHODS: Four hundred and three patients eligible for receiving i.v. chemotherapy for solid tumors were randomly assigned to implantation of a single type of port (Bard Port, Bard Inc., Salt Lake City, UT), through a percutaneous landmark access to the internal jugular, a ultrasound (US)-guided access to the subclavian or a surgical cut-down access through the cephalic vein at the deltoid-pectoralis groove. Early and late complications were prospectively recorded until removal of the device, patient's death or ending of the study. RESULTS: Four hundred and one patients (99.9%) were assessable: 132 with the internal jugular, 136 with the subclavian and 133 with the cephalic vein access. The median follow-up was 356.5 days (range 0-1087). No differences were found for early complication rate in the three groups {internal jugular: 0% [95% confidence interval (CI) 0.0% to 2.7%], subclavian: 0% (95% CI 0.0% to 2.7%), cephalic: 1.5% (95% CI 0.1% to 5.3%)}. US-guided subclavian insertion site had significantly lower failures (e.g. failed attempts to place the catheter in agreement with the original arm of randomization, P = 0.001). Infections occurred in one, three and one patients (internal jugular, subclavian and cephalic access, respectively, P = 0.464), whereas venous thrombosis was observed in 15, 8 and 11 patients (P = 0.272). CONCLUSIONS: Central venous insertion modality and sites had no impact on either early or late complication rates, but US-guided subclavian insertion showed the lowest proportion of failures.

Laboratory-based management of microbiological alerts: effects of an automated system on the surveillance and treatment of nosocomial infections in an oncology hospital.

BACKGROUND: Prevention and surveillance programs are key to contain Nosocomial Infections (Nis). At the European Institute of Oncology, surveillance based on ex-post data collection has been done since the inception of hospital activity; laboratory-based surveillance of microbiological alert was not standardized. This study describes the issues related to the recent introduction into the hospital routine of a laboratory-based automated surveillance system and its clinical impact on monitoring and treatment of Nis. METHODS: An interdisciplinary team defined the alerts and the actions to be taken in response; recipients of the alert messages were identified and software was programmed. Program features were created so their employment would generate a prompt notification of clinically critical results. After a training period, the program was introduced in the hospital routine. RESULTS: There were a total of 150 generated alerts; the main alert related to microorganisms requiring prompt patient isolation and/or public notification. Clinical use of the program was relevant in detection and immediate notification of Cytomegalovirus active infection in stem cell recipients and central venous catheter related candidemia: the prompt administration of adequate treatment was possible hours earlier compared to the previous approach. CONCLUSIONS: A laboratory-based automated surveillance system is effective in facilitating the management of Nis; its clinical employment also leads to important clinical advantages in patient care.

Spectroscopic magnetic resonance imaging of the brain: voxel localisation and tissue segmentation in the follow up of brain tumour.

The field of application of magnetic resonance spectroscopy (MRS) in biomedical research is expanding all the time and providing opportunities to investigate tissue metabolism and function. The data derived can be integrated with the information on tissue structure gained from conventional and non-conventional magnetic resonance imaging (MRI) techniques. Clinical MRS is also strongly expected to play an important role as a diagnostic tool. Essential for the future success of MRS as a clinical and research tool in biomedical sciences, both in vivo and in vitro, is the development of an accurate, biochemically relevant and physically consistent and reliable data analysis standard. Stable and well established analysis algorithms, in both the time and the frequency domain, are already available, as is free commercial software for implementing them. In this study, we propose an automatic algorithm that takes into account anatomical localisation, relative concentrations of white matter, grey matter, cerebrospinal fluid and signal abnormalities and inter-scan patient movement. The endpoint is the collection of a series of covariates that could be implemented in a multivariate analysis of covariance (MANCOVA) of the MRS data, as a tool for dealing with differences that may be ascribed to the anatomical variability of the subjects, to inaccuracies in the localisation of the voxel or slab, or to movement, rather than to the pathology under investigation. The aim was to develop an analysis procedure that can be consistently and reliably applied in the follow up of brain tumour. In this study, we demonstrate that the inclusion of such variables in the data analysis of quantitative MRS is fundamentally important (especially in view of the reduced accuracy typical of MRS measures compared to other MRI techniques), reducing the occurrence of false positives.

Lymphocyte subsets recovery following allogeneic bone marrow transplantation (BMT): CD4+ cell count and transplant-related mortality.

To assess the kinetics of lymphocyte subset recovery, 758 allografted patients were monitored by surface markers (CD3, CD4, CD8, CD56), with a 5-year follow-up. The donor was a matched sibling donor (MSD) (n=502) or an alternative donor (family mismatched or unrelated, AD) (n=256). The stem cell source was bone marrow for all patients. CD4+ cell recovery was influenced -- in univariate analysis -- by three factors: donor type, patient age and GvHD. This was not the case for CD8+ and CD56+ cells. The median CD4+ cell count on day +35 after HSCT was 86/mul. Patients achieving this CD4+ cell count had significantly lower transplant-related mortality (TRM) compared to patients who did not achieve this CD4+ cell count (20 vs 39%, P=0.00001), due to a lower risk of lethal infections (24 vs 47%, P=0.0003). In multivariate analysis MSD (RR 3.45, P=0.0001) and recipient age less than 16 years (RR 3.23, P=0.003) were significantly associated with a better CD4+ cell recovery. CD4+ counts on day +35 was predicted TRM (RR=1.97, P=0.0017) together with acute GvHD grade II-IV (RR 1.59, P=0.0097). No difference of TRM was observed for CD8+ and CD56+ cell counts.

Sex differences in human lymphocyte Na,K-ATPase as studied by labeled ouabain binding.

Lymphocytes Na,K-ATPase is a plasma membrane enzyme that is up-regulated under lymphocytes activation. It is also studied as a model of brain cells Na,K-ATPase. Data about sex-related specificities of the enzyme are not available. The binding of tritium-labelled ouabain to lymphocyte plasma membrane Na,K-ATPase was studied in healthy volunteers of both sexes. The binding interactions were expressed in terms of K(D) and B(Max). The first parameter is related to the affinity of ouabain for the enzyme whereas the second one is related to its density on the cell membrane. Distinct sex-related differences were found. Whereas in males there is a significant direct correlation between the parameters K(D) and B(Max), in females this is not present. However, in females there is a significantly lower K(D) in the 25-37 age range. The latter result probably reflects the expression of subunit variants giving a greater affinity for ouabain. This circumstance may be relevant both to lymphocytes' ability to be activated and to brain function, if one admits that lymphocyte Na,K-ATPase faithfully represents the brain-borne one.

Progenitor cells trapped in marrow filters can reduce GvHD and transplant mortality.

A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filter-discarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001-2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997-2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II-IV graft-versus-host disease (GvHD) (42 vs 15%, P=0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P=0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P=0.15). This study suggests that a significant proportion of LTC-IC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM.

RAR-mediated epigenetic control of the cytochrome P450 Cyp26a1 in embryocarcinoma cells.

Retinoic acid (RA) is a signaling molecule that plays a pivotal role in major cellular processes and vertebrate development. RA action is mediated by specialized transcription factors, the nuclear RA receptors (RARs), which regulate the transcription of genes containing a RA-responsive element (RARE). Here we demonstrate that the genes for the RA-receptor RARbeta2 and the cytochrome P450 RA-specific hydrolase Cyp26a1 involved in RA catabolism are coordinately regulated by RA. We found that both RARbeta2 and Cyp26a1 genes are epigenetically silenced in the absence of DNA methylation in RAC65, a P19 embryocarcinoma cell line derivative carrying a dominant-negative RARalpha mutant and resistant to the growth-inhibitory and differentiation effects of RA. In response to RA, RARbeta2 transcription is epigenetically regulated by RARalpha. Similarly, we found that Cyp26a1 transcription is epigenetically regulated by RARbeta2. Knocking down RARbeta2 transcription by RNA interference in wild-type P19 cells, with an intact RARalpha, induced Cyp26a1 transcriptional repression in the absence of DNA methylation. Concomitantly, cells developed RA resistance and did not undergo RA-induced neuron differentiation. Apparently, RARalpha, RARbeta2 and Cyp26a1 are components of a RA-regulated gene network. Factors affecting an upstream gene of the network can trigger repressive chromatin changes -- which are propagated in a domino fashion - at downstream genes of the network. This study also shows that chromatin inactivity, and consequent transcriptional silencing, can be achieved in the absence of DNA methylation.