A uniform method for the simultaneous blood group phenotyping of Fya , Fyb , Jka , Jkb , S, sÌ , P1, k applying lateral-flow technique.

BACKGROUND AND OBJECTIVES: A lateral flow assay for simultaneous blood group typing of ABO, RhD, C, E, c, e, Cw and K with stable end-point and without centrifugation is in routine use since several years (MDmulticard® ). The typing of extended phenotype parameters belonging to the Duffy, Kidd, MNSs blood group systems and others, however, has not yet been demonstrated for this technique. Reliable detection of Fyx , a weak Fyb phenotype with a pronounced quantitative reduction of the number of Fyb antigens on the erythrocyte surface, remains a weakness of current serological blood grouping techniques. MATERIAL AND METHODS: The performance characteristics of the following reagents were evaluated in donor and patient samples in lateral flow technology (MDmulticard® ): Anti-Fya , -Fyb , -Jka , -Jkb , -S, -s̅, -P1 and -k. The sensitivity to detect Fyx was in addition evaluated with Fyx positive samples, which had been preselected by MALDI-TOF MS-based genotyping. RESULTS: All results obtained with the MDmulticard® were in full accordance with those of the CE-certified reference products for all the eight reagent formulations used: Anti-Fya , -Fyb , -Jka , -Jkb , -S, -s̅, -P1 and -k. Also, all Fyx phenotypes of the selected population of 93 positive samples, originally identified by MALDI-TOF MS-based genotyping, were reliably detected by the lateral flow assay. CONCLUSION: Extended phenotype blood group parameters, including the serologically challenging Fyx phenotype, can be determined simultaneously, rapidly and accurately using the lateral flow (MDmulticard® ) technology, even in cases when IgG class antibodies are the only source of diagnostic antibodies.

The VKORC1 and CYP2C9 genotypes significantly effect Vitamin K antagonist dosing only in patients over the age of 20years.

INTRODUCTION: Given the qualitative differences in the role of VKORC1 and CYP2C9 polymorphisms in Vitamin K antagonists (VKA) dosing variation between adults and children, we were interested in determining at what age these polymorphism begin to play a more significant role. METHODS: A prospective cohort study of 190 patients aged 1-86years receiving VKA for treatment of venous thromboembolism. Blood samples were collected beyond the acute thrombotic event when patients were on stable targeted INR (2-3) for plasma testing and VKORC1/CYP2C9 genotyping. Patient demographics including VKA dose were collected. Simple and multiple linear regression was used to assess the relationship of VKA dose with polymorphisms and weight, adjusted for quality of anticoagulation (INR, D-Dimer), liver (AST, ALT) and renal function. RESULTS: In subjects 1-19years of age, weight explained 39.0% of dosing variation with VKORC1 and CYP2C9 playing a minor role. In contrast, in subjects 20-40years weight contributed 23%, VKORC1 44% and CYPC29 49% of the VKA dose variation. CONCLUSION: Until the age of 19, weight has a far greater effect on VKA dosing variation than VKORC1 and CYP2C9 polymorphisms. During the age of 20-40years, VKORC1 and CYP2C9 play a significant role.

Patient Blood Management in Europe: surveys on top indications for red blood cell use and Patient Blood Management organization and activities in seven European university hospitals.

BACKGROUND AND OBJECTIVES: Patient Blood Management (PBM) in Europe is a working group of the European Blood Alliance with the initial objective to identify the starting position of the participating hospitals regarding PBM for benchmarking purposes, and to derive good practices in PBM from the experience and expertise in the participating teams with the further aim of implementing and strengthening these practices in the participating hospitals. METHODS: We conducted two surveys in seven university hospitals in Europe: Survey on top indications for red blood cell use regarding usage of red blood cells during 1 week and Survey on PBM organization and activities. RESULTS: A total of 3320 units of red blood cells were transfused in 1 week at the seven hospitals. Overall, 61% of red cell units were transfused to medical patients and 36% to surgical patients, although there was much variation between hospitals. The organization and activities of PBM in the seven hospitals were variable, but there was a common focus on optimizing the treatment of bleeding patients, monitoring the use of blood components and treatment of preoperative anaemia. CONCLUSION: Although the seven hospitals provide a similar range of clinical services, there was variation in transfusion rates between them. Further, there was variable implementation of PBM activities and monitoring of transfusion practice. These findings provide a baseline to develop joint action plans to further implement and strengthen PBM across a number of hospitals in Europe.

[Cost analysis of patient blood management]. / Kostenanalyse eines Patient-Blood-Management-Konzepts.

BACKGROUND: Patient blood management (PBM) is a multidisciplinary approach focusing on the diagnosis and treatment of preoperative anaemia, the minimisation of blood loss, and the optimisation of the patient-specific anaemia reserve to improve clinical outcomes. Economic aspects of PBM have not yet been sufficiently analysed. OBJECTIVES: The aim of this study is to analyse the costs associated with the clinical principles of PBM and the project costs associated with the implementation of a PBM program from an institutional perspective. MATERIALS AND METHODS: Patient-related costs of materials and services were analysed at the University Hospital Frankfurt for 2013. Personnel costs of all major processes were quantified based on the time required to perform each step. Furthermore, general project costs of the implementation phase were determined. RESULTS: Direct costs of transfusing a single unit of red blood cells can be calculated to a minimum of €147.43. PBM-associated costs varied depending on individual patient requirements. The following costs per patient were calculated: diagnosis of preoperative anaemia €48.69-123.88; treatment of preoperative anaemia (including iron-deficiency anaemia and megaloblastic anaemia) €12.61-127.99; minimising perioperative blood loss (including point-of-care diagnostics, coagulation management and cell salvage) €3.39-1,901.81; and costs associated with the optimisation of the tolerance to anaemia (including patient monitoring and volume therapy) €28.62. General project costs associated with the implementation of PBM were €24,998.24. CONCLUSIONS: PBM combines various alternatives to the transfusion of red blood cells and improves clinical outcome. Costs of PBM vary from institution to institution and depend on the extent to which different aspects of PBM have been implemented. The quantification of costs associated with PBM is essential in order to assess the economic impact of PBM, and thereby, to efficiently re-allocate health care resources. Costs were determined at a single university hospital. Thus, further analyses of both the costs of transfusion and the costs of PBM-principles will be necessary to evaluate the cost-effectiveness of PBM.

Pathogen-reduced Ebola virus convalescent plasma: first steps towards standardization of manufacturing and quality control including assessment of Ebola-specific neutralizing antibodies.

BACKGROUND: Ebola virus disease is a public health emergency of international concern, and enormous efforts are being made in the development of vaccines and therapies. Ebola virus convalescent plasma is a promising anti-infective treatment of Ebola virus disease. Therefore, we developed and implemented a pathogen-reduced Ebola virus convalescent plasma concept in accordance with national, European and global regulatory framework. MATERIALS AND METHODS: Ebola virus convalescent plasma manufacture and distribution was managed by a collection centre, two medical centres and an expert group from the European Blood Alliance. Ebola virus convalescent plasma was collected twice with an interval of 61 days from a donor recovering from Ebola virus disease in Germany. After pathogen reduction, the plasma was analysed for Ebola virus-specific immunoglobulin G (IgG) antibodies and its Ebola virus neutralizing activity. RESULTS: Convalescent plasma could be collected without adverse events. Anti-Ebola virus IgG titres and Ebola-specific neutralizing antibodies in convalescent plasma were only slightly reduced after pathogen reduction treatment with S59 amotosalen/UVA. A patient in Italy with Ebola virus disease was treated with convalescent plasma without apparent adverse effects. DISCUSSION: As proof of principle, we describe a concept and practical implementation of pathogen-reduced Ebola virus convalescent plasma manufacture, quality control and its clinical application to an Ebola virus disease patient.

[Patient blood management: Current state of the literature]. / Patient-blood-Management : Stand der aktuellen Literatur.

BACKGROUND: Preoperative anemia has a prevalence of approximately 30% and is one of the strongest predictors of perioperative red blood cell (RBC) transfusion. It is rarely treated although it is an independent risk factor for the occurrence of postoperative complications. Additionally, the high variability in the worldwide usage of RBC transfusions is alarming. Due to these serious deficits in patient care, in 2011 the World Health Organization recommended the implementation of a patient blood management (PBM). OBJECTIVES: This article provides information about PBM as a multidimensional and interdisciplinary approach. MATERIAL AND METHODS: A selective literature search was carried out in the Medline and Cochrane library databases including consideration of national and international guidelines. RESULTS: A PBM promotes the medically and ethically appropriate use of all available resources, techniques and materials in favor of an optimized perioperative patient care. Patients' own resources should be specifically protected, strengthened and used and include (i) diagnosis and therapy of preoperative anemia, (ii) minimizing perioperative blood loss, (iii) blood-conserving surgical techniques, (iv) restriction of diagnostic blood sampling, (v) utilization of individual anemia tolerance, (vi) optimal coagulation and hemotherapy concepts and (vii) guideline-based, rational indications for the use of RBC transfusions. CONCLUSION: A PBM should be advocated as an incentive to evaluate and critically optimize local conditions. An individual, interdisciplinarily structured bundle of different PBM measures has great potential to optimize the quality of patient care and to make it safer.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

Arterial thrombosis in homozygous antithrombin deficiency.

UNLABELLED: Antithrombin (AT), a serin protease inhibitor (serpin) produced in the liver, inhibits mainly thrombin and factor Xa. Antithrombin deficiency (AD) is associated with a higher incidence of thrombosis. CASE REPORT: We report a newborn with uncomplicated birth in the 40+5 week of gestation and postnatal appearance of a reticular, livide haematoma on the right upper arm and a tonic clonic epileptic seizure. Clinical examination revealed weak pulses in the A. radialis and ulnaris. MRI scan showed a large thrombus in the A. carotis interna and externa with large cerebral infarction and a thrombus in the A. subclavia. Laboratory work up showed elevated D-dimers and antithrombin levels <20% (lowest 15%), age-related values for protein C, protein S, plasminogen, and no other inherited thrombophilia. THERAPY: We started anticoagulation with unfractionated heparin intravenously (aPTT: 50-60 s) and under suspicion of an AD the substitution of AT (70 U/kg body weight). In course of time we changed anticoagulation to low molecular weight heparin (Anti Xa 0.6-0.8 U/ml) and substitution of 250 E/kg AT every second day. In the molecular work up we found a homozygous missense mutation in exon 2 of SERPINC1 gene (type "Budapest 3"). Molecular analysis showed also heterozygous mutations in both parents and a homozygous mutation in the asymptomatic brother aged three years. At age of six months we changed the anticoagulation to coumadin (INR 2.5-3.5). Anticoagulation with coumadin was also started in the brother. DISCUSSION: Hereditary AD is associated with an increased risk of thrombosis. The homozygous status mainly leads to intrauterine fetal loss or the occurrence of peri- and postnatal thrombosis. Therapy consists in the substitution of AT and a lifelong anticoagulation with vitamin K antagonists also in asymptomatic patients.

Apheresis product identification in the transplant center: development of point-of-care protocols for extended blood typing of stem cell apheresis products.

Transfusion of the 'wrong' stem cell product would almost inevitably be lethal, yet assays to confirm the contents of the product bag, except by checking labels and paperwork, are lacking. To increase the likelihood that a product mix-up would be detected in the transplant center, we developed a simple protocol for extended blood typing and hence, for confirmation of donor/product identity, on a tube segment. Apheresis samples were applied, directly or after erythrocyte enrichment, to commercially available blood typing assays, including lateral flow cards and gel agglutination cards. Without sample modification, low hematocrit and high leukocyte count obviated definitive blood typing. Using the most simple erythrocyte enrichment protocol, that is, centrifugation, reliable blood group analysis became possible with either assay. Other, more cumbersome pre-analytical protocols were also successful but provided no advantage. The preferred method was validated on 100 samples; ABD was correctly identified in 100% of cases. Of the other Rh Ags, all except two 'small e', in both cases in heterozygous individuals, were detected; there were no false positives. A simple, inexpensive point-of-care assay for extended blood typing of apheresis products is available, which can reduce the fatal risk of administering the wrong stem cell product.

Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment.

BACKGROUND: Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. OBJECTIVES: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. PATIENTS/METHODS: Six hundred and twenty-six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. RESULTS: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26→Thr, His28→Gln, Asp36→Gly, Ser52→Trp, Ser56→Phe, Trp59→Leu, Trp59→Cys, Val66→Gly, Gly71→Ala, Asn77→Ser, Asn77→Tyr, Ile123→Asn, and Tyr139→His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29→Leu, Asp36→Tyr, and Val66→Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non-VKORC1*2 and wild-type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane-lumen interface. CONCLUSIONS: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.

Association between phenotype and genotype in carriers of haemophilia A.

Female carriers of haemophilia might suffer from increased bleeding tendency therefore the assessment of the bleeding risk is very important for improving care. This single-centre study documents the occurrence of bleedings in 46 carriers of haemophilia A including bleeding after tooth extraction (77%), easy bruising (67%), postsurgical bleeding (61%), menorrhagia (50%) or prolonged postpartum bleeding (43%). The F8 gene mutation of all 46 carriers (median age: 36.5 years, 15-80 years; mean FVIII:C activity: 59 ± 24.45%; normal range: 64-167%) was determined, and family history of haemophilia was recorded. For analysis, the bleeding tendency of the carriers was differentiated by severity into three groups. There was no statistically significant difference of FVIII:C between these groups. However, a correlation was found between the severity of bleeding tendency and the type of F8 gene mutation (P < 0.05) as well as the severity of haemophilia in affected male relatives (P < 0.0005). Results show that even carriers with a FVIII:C activity as high as 50-60% are at increased risk of bleeding. Incidence and intensity of bleeding symptoms of haemophilia A carriers are high and correlated with the phenotype of the male haemophilic relative and the underlying F8 gene mutation.

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53.

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.

Cerebral blood flow and glucose metabolism in multi-infarct-dementia related to primary antiphospholipid antibody syndrome.

The primary antiphospholipid antibody syndrome (PAPS) has been described in patients with a history of fetal loss, thrombocytopenia and arterial or venous thrombosis. In PAPS, a prothrombotic state is mediated by antiphospholipid antibodies (aPLs) leading to disseminated thromboembolic vascular occlusion. Today, the presence of aPLs in the serum is considered as a distinct risk factor for recurrent stroke in young adults. Some PAPS patients develop a multi-infarct-syndrome with a stepwise decline of higher cortical functions. We report on a 55-year-old man suffering from progressive dementia and PAPS, in whom cerebral glucose metabolism and blood flow were examined by positron emission tomography (PET). Cerebral atrophy and moderate signs of leukaraiosis were detected in magnetic resonance imaging (MRI), whereas the PET scans showed a considerable diffuse impairment of cortical glucose metabolism combined with a reduced cerebral perfusion in the arterial border zones. These findings indicate that PAPS-associated vascular dementia is accompanied by a cortical neuronal loss, presumably caused by a small-vessel disease with immune-mediated intravascular thrombosis. This case shows that pathological findings in PAPS are congruent to cerebral changes of metabolism and blood flow in systemic lupus erythematosus (SLE).

Growth inhibition of cervical cancer cells by the human retinoic acid receptor beta gene.

Transcription of the retinoic receptor beta (RARbeta) gene is activated in a ligand-dependent manner by the retinoic acid receptor alpha. Reduced RARbeta gene expression and loss of ligand inducibility are frequently observed in human carcinoma cells indicating that such alterations might contribute to carcinogenesis. In this study we have analyzed the influence of RARbeta on cervical cancer cell growth. Transfection of HeLa cells with RARbeta expression plasmids resulted in reduced clonal cell growth in the presence of retinoic acid (RA). RA-induced growth inhibition in HeLa x fibroblast hybrid cells was partially relieved by a dominant-negative RARbeta mutant. HeLa clones stably expressing a RARbeta transgene under control of the human beta-actin promoter [HeLa(RARbeta)] were established and analyzed for transgene-mediated growth alterations in vitro and in vivo. Anchorage-independent growth of the HeLa(RARbeta) lines was indistinguishable from that of control cells in the absence of RA, but strongly impaired after RA treatment. Reduced tumor growth of HeLa(RARbeta) clones was associated with high RARbeta protein levels. Somatic cell fusion experiments revealed that the loss of ligand inducibility of RARbeta gene expression in HeLa cells cannot be complemented by fusion with other cervical cancer cell lines. Our data indicate, firstly, that RARbeta is a negative regulator of tumor cell growth and, secondly, that cancer-associated defects in RARbeta gene expression are caused by stable, non-complementable silencing mechanisms.

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.

Oncogenic potential of cyclin E in T-cell lymphomagenesis in transgenic mice: evidence for cooperation between cyclin E and Ras but not Myc.

To study the oncogenic activity of cyclin E in an in vivo system we generated transgenic mice expressing high levels of cyclin E in T-lymphocytes by using a construct containing the CD2 locus control region. These animals were neither predisposed to develop any tumors spontaneously nor showed an increased incidence when crossbred with Emu L-myc transgenic mice but developed hyperplasia in peripheral lymphoid organs at later age with an incidence of 27%. When treated with the DNA methylating carcinogen N-methylnitrosourea (MNU) that provokes the development of T-cell lymphomas, CD2-cyclin E transgenic animals came down with T-cell neoplasia showing a significant higher incidence (54%) than normal non transgenic controls (31%). In one of eight tumors that arose in normal MNU treated mice we could find an expected activating point mutation in the Ki-ras gene (12.5%). In contrast, the same mutation occurred in five of 16 tumors from CD2-cyclin E transgenic mice (31.2%). Whereas cyclin E overexpression alone did not lead to an increased CDK2 activity we observed in all tumors that emerged from either MNU treated normal mice or treated CD2-cyclin E transgenics a downregulation of p27KIP1 and a higher histone H1 kinase activity in CDK2 immunoprecipitates compared to normal tissue. These findings demonstrate that high level expression of cyclin E can predispose T-cells for hyperplasia and malignant transformation. However, the results also suggest that this activity of cyclin E is manifest only when other cooperating oncogenes in particular ras genes are present and activated. This would be consistent with our previous finding that cyclin E and Ha-Ras cooperate in focus formation assays in rat embryo fibroblasts.

Investigation of the cell cycle regulation of cdk3-associated kinase activity and the role of cdk3 in proliferation and transformation.

The G1-S transition in mammalian cells has been demonstrated to require the cyclin-dependent kinases cdk2, cdk3 and cdk4/6. Here we show that a novel kinase activity associated with cdk3 fluctuates throughout the cell cycle differently from the expression of cyclin D1-, E- and A-associated kinase activities. Cdk3 kinase activity is neither affected by p16 (in contrast to cdk4/6) nor by E2F-1 (in contrast to cdk2), but is downregulated upon transient p27 expression. We found cdk3 to bind to p21 and p27. We provide evidence that p27 could be involved in the regulation of the cell cycle fluctuation of cdk3 activity: cdk3 protein does not fluctuate and interaction of cdk3 with p27, but not with p21, is lost when cdk3 kinase becomes active during the cell cycle. In Myc-overexpressing cells, but not in normal Ratl cells, constitutive ectopic expression of cdk3 induces specific upregulation of cdk3-associated kinase activity that is still cell cycle phase dependent. Ectopic cdk3, but not cdk2, enhances Myc-induced proliferation and anchorage-independent growth associated with Myc activation, without effects on cyclin D1, E and A protein expression or kinase activities. High levels of cdk3 in Myc-overexpressing cells trigger up- and deregulation of E2F-dependent transcription without inducing the E2F-DNA binding capacity. In contrast to all other studied positive G regulators, cdk3 is unable to cooperate with ras in fibroblast transformation suggesting a function of cdk3 in G1 progression that is different from cyclin D- or E-associated kinase activities. Our data provide first insights into the regulation of cdk3-associated kinase activity and suggest a model how cdk3 participates in the regulation of the G1-S transition.

Sulindac sulfide inhibits Ras signaling.

The non-steroidal anti-inflammatory drug sulindac is used in cancer prevention and therapy, but the molecular aspects of its anti-tumor effect remain unresolved. In vivo the prodrug sulindac, is converted into the metabolite sulindac sulfide. We found that sulindac sulfide strongly inhibits Ras induced malignant transformation and Ras/Raf dependent transactivation. Sulindac sulfide decreases the Ras induced activation of its main effector, the c-Raf-1 kinase. In vitro sulindac sulfide directly binds to the Ras gene product p21ras in a non-covalent manner. Moreover, we can show that sulindac sulfide inhibits the interaction of p21ras with the p21ras binding domain of the Raf protein. In addition, sulindac sulfide can impair the nucleotide exchange on p21ras by CDC25 as well as the acceleration of the p21ras GTPase reaction by p120GAP. Due to its action at the most critical site in Ras signaling we propose sulindac sulfide as a lead compound in the search for novel anti-cancer drugs which directly inhibit Ras mediated cell proliferation and malignant transformation.