Adjuvant chemotherapy for resected duodenal adenocarcinoma: a case-matched analysis in nation wide cohort.

BACKGROUND: Duodenal adenocarcinoma (DA) is a rare tumor for which survival data on adjuvant chemotherapy in patients after surgical treatment are unclear. This case-matched study in a nationwide cohort aims to investigate the benefit of adjuvant chemotherapy for patients with resectable DA on overall survival. METHODS: All patients diagnosed with DA and intestinal type periampullary adenocarcinoma (PVA) in the Netherlands between 2000 and 2015 were included (n = 1316). Patients with disease stages II and III who underwent resection and adjuvant chemotherapy were matched (1:2), based on identified covariates associated with OS, with patients who underwent surgery alone. Overall survival was compared using Kaplan-Meier estimates. RESULTS: The median OS was 49.9 months in patients who underwent curative resection (n = 649). Univariate and multivariate analysis showed a significant influence of age, lymph node involvement, and T- stage on survival. The group of patients receiving adjuvant treatment consisted of 43 patients and the non-adjuvant group of 83 case-matched patients. The median OS of the complete matched cohort (n = 126) was 26.9 months. No statistically significant survival benefit was found for the adjuvant group as compared to the group treated with surgery alone (median OS = 34.4 months and 23.0 months, p = 0.20). CONCLUSION: This population-based, case-matched analysis demonstrates no statistically significant survival benefit for adjuvant chemotherapy after curative resection in stages II and III patients. Future studies with specified treatment regimens as well as thorough stratification for prognostic factors will be required in order to more definitively determine the role of adjuvant therapy.

Resting energy expenditure in children at risk of hypothalamic dysfunction.

Objective: Children with suprasellar brain damage are at risk of hypothalamic dysfunction (HD). HD may lead to decreased resting energy expenditure (REE). Decreased REE, however, is not present in all children with HD. Our aim was to assess which children suspect for HD have low REE, and its association with clinical severity of HD or radiological hypothalamic damage. Patients and methods: A retrospective cohort study was performed. Measured REE (mREE) of children at risk of HD was compared to predicted REE (pREE). Low REE was defined as mREE <90% of predicted. The mREE/pREE quotient was associated to a clinical score for HD symptoms and to radiological hypothalamic damage. Results: In total, 67 children at risk of HD (96% brain tumor diagnosis) with a mean BMI SDS of +2.3 ± 1.0 were included. Of these, 45 (67.2%) had low mREE. Children with severe HD had a significant lower mean mREE/pREE quotient compared to children with no, mild, or moderate HD. Mean mREE/pREE quotient of children with posterior hypothalamic damage was significantly lower compared to children with no or anterior damage. Tumor progression or tumor recurrence, severe clinical HD, and panhypopituitarism with diabetes insipidus (DI) were significant risk factors for reduced REE. Conclusion: REE may be lowered in children with hypothalamic damage and is associated to the degree of clinical HD. REE is, however, not lowered in all children suspect for HD. For children with mild or moderate clinical HD symptoms, REE measurements may be useful to distinguish between those who may benefit from obesity treatment that increases REE from those who would be better helped using other obesity interventions.

Cerebral Phospho-Tau Acts Synergistically with Soluble Aß42 Leading to Mild Cognitive Impairment in AAV-AD Rats.

BACKGROUND: Alzheimer's disease (AD) is a continuum of events beginning with an increase in brain soluble Aß42 followed by the appearance of hyperphosphorylated tau (P-tau, asymptomatic stage). Mild Cognitive Impairment (MCI) then appears (prodromal stage). However, the individual contribution of these two soluble proteins in the onset of the first cognitive symptoms remains unclear. OBJECTIVES: We sought to understand the specific impact of p-tau on the development of MCI in the AAV-AD rat model, a model of late-onset Alzheimer's disease (LOAD) predementia. METHODS: We specifically reduced the phosphorylation level of tau while leaving Aß42 levels unchanged using a DYRK1A protein kinase inhibitor, Leucettine L41, in an adeno-associated virus-based Alzheimer's disease (AAV-AD) rat model. Leucettine L41 was administered by intraperitoneal injection at 20 mg/kg per day in AAV-AD rats from 9 (late asymptomatic phase) to 10 (prodromal phase) months of age. RESULTS: Decreased soluble forms of P-tau induced by chronic administration of Leucettine L41 did not change soluble Aß42 levels but prevented MCI onset in 10-month-old AAV-AD rats. CONCLUSIONS: The present study argues that P-tau is required to induce the development of MCI. Consistent with our previous findings that soluble Aß42 is also required for MCI onset, the data obtained in the AAV-AD rat model confirm that the transition from the asymptomatic to the prodromal stage may be caused by the combined presence of both soluble brain forms of Aß42 and p-tau, suggesting that the development of MCI may be the consequence of their synergistic action.

Extensive diagnostic work-up for patients with carcinoma of unknown primary.

Patients with carcinoma of unknown primary (CUP) present with metastatic disease without an identified primary tumour. The unknown site of origin makes the diagnostic work-up and treatment challenging. Since little information is available regarding diagnostic work-up and treatment in daily practice, we collected and analysed these in a patient cohort with regard to the recommendations of the national CUP guideline. Data of 161 patients diagnosed with CUP in 2014 or 2015 were extracted from the Netherlands Cancer Registry (NCR) and supplemented with diagnostic work-up information from patient files and analysed. Patients underwent an average of five imaging studies during the diagnostic phase (range 1-17). From the tests as recommended in the national guideline on CUP, a chest X-ray was most commonly performed (73%), whereas a PET-CT was done in one out of four patients (24%). Biopsies were taken in 86% of the study population, with Cytokeratin 7 being the most frequently tested histopathological marker (73%). Less than half of patients received therapy (42%). CUP patients undergo extensive diagnostic work-up. The performance status did not influence the extent of the diagnostic work-up in CUP patients, but it was an important factor for receiving treatment.

Crossing borders: A systematic review with quantitative analysis of genetic mutations of carcinomas of the biliary tract.

Biliary tract carcinoma (BTC) comprises gallbladder and intra-/extrahepatic cholangiocarcinoma (GBC, ICC, EHC), which are currently classified by anatomical origin. Better understanding of the mutational profile of BTCs might refine classification and improve treatment. We performed a systematic review of studies reporting on mutational profiling of BTC. We included articles reporting on whole-exome/whole-genome-sequencing (WES/WGS) and targeted sequencing (TS) of BTC, published between 2000-2017. Pooled mutation proportions were calculated, stratified by anatomical region and sequencing technique. A total of 25 studies with 1806 patients were included. Overall, TP53 was the most commonly mutated gene in BTC. GBC was associated with mutations in PFKFB3, PLXN2 and PGAP1. Mutations in IDH1, IDH2 and FGFR fusions almost exclusively occurred in ICC patients. Mutations in APC, GNAS and TGFBR2 occurred exclusively in EHC patients. In conclusion, subtypes of BTCs exhibit minor differences in mutational profile, which is likely influenced by the cell of origin.

Monitoring cognitive change in multiple sclerosis using a computerized cognitive battery.

BACKGROUND: Cognitive monitoring that can detect short-term change in multiple sclerosis is challenging. Computerized cognitive batteries such as the CogState Brief Battery can rapidly assess commonly affected cognitive domains. OBJECTIVES: The purpose of this study was to establish the acceptability and sensitivity of the CogState Brief Battery in multiple sclerosis patients compared to controls. We compared the sensitivity of the CogState Brief Battery to that of the Paced Auditory Serial Addition Test over 12 months. METHODS: Demographics, Expanded Disability Status Scale scores, depression and anxiety scores were compared with CogState Brief Battery and Paced Auditory Serial Addition Test performances of 51 patients with relapsing-remitting multiple sclerosis, 19 with secondary progressive multiple sclerosis and 40 healthy controls. Longitudinal data in 37 relapsing-remitting multiple sclerosis patients were evaluated using linear mixed models. RESULTS: Both the CogState Brief Battery and the Paced Auditory Serial Addition Test discriminated between multiple sclerosis and healthy controls at baseline (p<0.001). CogState Brief Battery tasks were more acceptable and caused less anxiety than the Paced Auditory Serial Addition Test (p<0.001). In relapsing-remitting multiple sclerosis patients, reaction time slowed over 12 months (p<0.001) for the CogState Brief Battery Detection (mean change -34.23 ms) and Identification (-25.31 ms) tasks. Paced Auditory Serial Addition Test scores did not change over this time. CONCLUSIONS: The CogState Brief Battery is highly acceptable and better able to detect cognitive change than the Paced Auditory Serial Addition Test. The CogState Brief Battery could potentially be used as a practical cognitive monitoring tool in the multiple sclerosis clinic setting.

Ten- to 15-year results of the Oxford Phase III mobile unicompartmental knee arthroplasty: a prospective study from a non-designer group.

AIMS: The interest in unicompartmental knee arthroplasty (UKA) for medial osteoarthritis has increased rapidly but the long-term follow-up of the Oxford UKAs has yet to be analysed in non-designer centres. We have examined our ten- to 15-year clinical and radiological follow-up data for the Oxford Phase III UKAs. PATIENTS AND METHODS: Between January 1999 and January 2005 a total of 138 consecutive Oxford Phase III arthroplasties were performed by a single surgeon in 129 patients for medial compartment osteoarthritis (71 right and 67 left knees, mean age 72.0 years (47 to 91), mean body mass index 28.2 (20.7 to 52.2)). Both clinical data and radiographs were prospectively recorded and obtained at intervals. Of the 129 patients, 32 patients (32 knees) died, ten patients (12 knees) were not able to take part in the final clinical and radiological assessment due to physical and mental conditions, but via telephone interview it was confirmed that none of these ten patients (12 knees) had a revision of the knee arthroplasty. One patient (two knees) was lost to follow-up. RESULTS: The mean follow-up was 11.7 years (10 to 15). A total of 11 knees (8%) were revised. The survival at 15 years with revision for any reason as the endpoint was 90.6% (95% confidence interval (CI) 85.2 to 96.0) and revision related to the prosthesis was 99.3% (95% CI 97.9 to 100). The mean total Knee Society Score was 47 (0 to 80) pre-operatively and 81 (30 to 100) at latest follow-up. The mean Oxford Knee Score was 19 (12 to 40) pre-operatively and 42 (28 to 55) at final follow-up. Radiolucency beneath the tibial component occurred in 22 of 81 prostheses (27.2%) without evidence of loosening. CONCLUSION: This study supports the use of UKA in medial compartment osteoarthritis with excellent long-term functional and radiological outcomes with an excellent 15-year survival rate. Cite this article: Bone Joint J 2016;98-B(10 Suppl B):41-7.

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism.

BACKGROUND AND PURPOSE: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. EXPERIMENTAL APPROACH: Human CF airway epithelial cells were treated with roscovitine 100 µM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. KEY RESULTS: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. CONCLUSIONS AND IMPLICATIONS: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.

Metrics and textural features of MRI diffusion to improve classification of pediatric posterior fossa tumors.

BACKGROUND AND PURPOSE: Qualitative radiologic MR imaging review affords limited differentiation among types of pediatric posterior fossa brain tumors and cannot detect histologic or molecular subtypes, which could help to stratify treatment. This study aimed to improve current posterior fossa discrimination of histologic tumor type by using support vector machine classifiers on quantitative MR imaging features. MATERIALS AND METHODS: This retrospective study included preoperative MRI in 40 children with posterior fossa tumors (17 medulloblastomas, 16 pilocytic astrocytomas, and 7 ependymomas). Shape, histogram, and textural features were computed from contrast-enhanced T2WI and T1WI and diffusivity (ADC) maps. Combinations of features were used to train tumor-type-specific classifiers for medulloblastoma, pilocytic astrocytoma, and ependymoma types in separation and as a joint posterior fossa classifier. A tumor-subtype classifier was also produced for classic medulloblastoma. The performance of different classifiers was assessed and compared by using randomly selected subsets of training and test data. RESULTS: ADC histogram features (25th and 75th percentiles and skewness) yielded the best classification of tumor type (on average >95.8% of medulloblastomas, >96.9% of pilocytic astrocytomas, and >94.3% of ependymomas by using 8 training samples). The resulting joint posterior fossa classifier correctly assigned >91.4% of the posterior fossa tumors. For subtype classification, 89.4% of classic medulloblastomas were correctly classified on the basis of ADC texture features extracted from the Gray-Level Co-Occurence Matrix. CONCLUSIONS: Support vector machine-based classifiers using ADC histogram features yielded very good discrimination among pediatric posterior fossa tumor types, and ADC textural features show promise for further subtype discrimination. These findings suggest an added diagnostic value of quantitative feature analysis of diffusion MR imaging in pediatric neuro-oncology.

CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells.

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

Constitutive TLR4 signalling in intestinal epithelium reduces tumor load by increasing apoptosis in APC(Min/+) mice.

The microbial pattern-recognizing Toll-like receptors (TLRs) are major signal transducers known to shape and influence the postnatal maturation of host intestinal epithelium. Perturbations in this intricate host-microbe cross-talk have been reported to be associated with uncontrolled epithelial cell growth and thus potential cancer development by mechanisms which are largely unknown. We therefore generated transgenic mice carrying a constitutively active TLR4 (CD4-TLR4) linked to an intestinal epithelial cell-specific promoter. Ex vivo analysis of transgenic crypt-villus organoid cultures revealed an increased proliferative capacity and a lowered cyclooxygenase 2 (Cox-2) expression in these organoids compared with wild-type control cultures. Introducing the CD4-TLR4 transgene into APC(Min/+) mice (CD4-TLR4-APC(Min/+)), a model of colorectal carcinoma, resulted in a dramatic drop in tumor load as compared with control APC(Min/+) mice. Intestinal tumors from CD4-TLR4-APC(Min/+) mice displayed reduced Cox-2 protein, elevated interferon ß expression and increased caspase-3 activity, which correlated with increased apoptosis in vivo. Thus, our data reveal that host microbiota-mediated signal transduction via TLR4 in intestinal epithelial cells is far more complex than what is previously reported.

An exercise-based physical therapy program for patients with patellar tendinopathy after platelet-rich plasma injection.

OBJECTIVES: To describe a post platelet-rich plasma (PRP) injection, exercise-based physical therapy program, investigate feasibility and report the first results of patellar tendinopathy patients treated with PRP injection combined with the physical therapy program. STUDY DESIGN: Case-series. SETTING: A PRP injection followed by a physical therapy program seems promising for the treatment of patellar tendinopathy. However, descriptions of physical therapy programs are often limited and incomplete. PARTICIPANTS: Five patellar tendinopathy patients (six tendons) in the degenerative phase. MAIN OUTCOME MEASURE: VISA-P score. RESULTS: Muscle strength, endurance, power and retraining sport-specific function form the basis for the physical therapy program aiming to improve the load capacity of the knee. The program is characterised by gradually increasing intensity and difficulty of exercises. Five of the six tendons showed an improvement of at least 30 points on the VISA-P after 26 weeks. CONCLUSIONS: This study extensively describes, based on current knowledge, a physical therapy program after PRP injection for patellar tendinopathy patients. The combination treatment reported in this study is feasible and seems to be promising for patients in the late/degenerative phase of patellar tendinopathy.

Influence of prenatal organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length.

BACKGROUND: Prenatal exposure to endocrine disruptors, like organohalogen compounds (OHCs), might be responsible for the increased aberrations in human male sexual development (hypospadias, cryptorchidism, testicular cancer and fall in sperm count) observed over the past decades. This development is established during fetal life, and reflected in sex hormone levels, testes volume and penile length post-partum. The present study investigates the correlation between prenatal OHC levels and male sexual development outcomes. METHODS AND RESULTS: Levels of eight neutral [2,2'-bis-(4-chlorophenyl)-1,1'-dichloroethene (4,4'-DDE), 2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4'-tetrabromodiphenyl ether (BDE)-47, -99, -100, -153, -154 and 1,2,5,6,9,10-hexabromocyclododecane, HBCDD] and four phenolic [(pentachlorophenol (PCP), 4OH-CB-107 (4-hydroxy-2,3,3',4',5-pentachlorobiphenyl), -146 and -187)] OHCs were determined in 55 maternal serum samples taken at 35 weeks of pregnancy. Eight sex development-related hormones [testosterone, free testosterone, sex hormone-binding globulin (SHBG); LH, FSH, estradiol (E(2)), free E(2) (FE(2)) and inhibin B (InhB)] were determined in their sons at 3 months of age, and testes volume and penile length at 3 and 18 months of age. The following prenatal OHC levels correlated significantly with sex hormone levels: PCP with SHBG and InhB (ρ = 0.30 and -0.43, respectively), 4OH-CB-107 with testosterone (ρ = 0.31) and BDE-154 with FE(2), E(2) and InhB (ρ = 0.49, 0.54 and 0.34, respectively). BDE-154 levels correlated positively with testes volume at 18 months of age (ρ = 0.34). CONCLUSIONS: Prenatal OHC exposure is correlated with aspects of sexual development outcome in boys up to 18 months of age.

A novel interaction between HER2/neu and cyclin E in breast cancer.

HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. As both are involved in cell cycle regulation we analyzed whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P<0.0001). In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 small interfering RNA or treatment with trastuzumab. Cyclin E expression levels were determined by western blot analysis, and functional effects analyzed using kinase assays, MTT assays were used to assess cell viability as a marker of proliferation and fluorescence-activated cell sorting analysis was used to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional consequences, including decreased cyclin E-associated kinase activity and decreased proliferation, because of increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies show that HER2-mediated signaling effects LMW cyclin E expression, which in turn deregulates the cell cycle. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.

CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases.

Among the ten pharmacological inhibitors of cyclin-dependent kinases (CDKs) currently in clinical trials, the purine roscovitine (CYC202, Seliciclib) is undergoing phase 2 trials against non-small-cell lung and nasopharyngeal cancers. An extensive medicinal chemistry study, designed to generate more potent analogues of roscovitine, led to the identification of an optimal substitution at the N6 position (compound CR8). An extensive selectivity study (108 kinases) highlights the exquisite selectivity of CR8 for CDK1/2/3/5/7/9. CR8 was 2- to 4-fold more potent than (R)-roscovitine at inhibiting these kinases. Cocrystal structures of (R)-CR8 and (R)-roscovitine with pCDK2/cyclin A showed that both inhibitors adopt essentially identical positions. The cellular effects of CR8 and (R)-roscovitine were investigated in human neuroblastoma SH-SY5Y cells. CR8 inhibited the phosphorylation of CDK1 and 9 substrates, with a 25-50 times higher potency compared to (R)-roscovitine. CR8 was consistently more potent than (R)-roscovitine at inducing apoptotic cell death parameters: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction (40-fold), lactate dehydrogenase release (35-fold), caspases activation (68-fold) and poly-(ADP-ribose)polymerase cleavage (50-fold). This improved cell death-inducing activity of CR8 over (R)-roscovitine was observed in 25 different cell lines. Altogether these results show that second-generation analogues of (R)-roscovitine can be designed with improved antitumor potential.

CDK/GSK-3 inhibitors as therapeutic agents for parenchymal renal diseases.

Drug discovery to lessen the burden of chronic renal failure and end-stage renal disease remains a principle goal of translational research in nephrology. In this review, we provide an overview of the current development of small molecule cyclin-dependent kinase (CDK)/glycogen synthase kinase-3 (GSK-3) inhibitors as therapeutic agents for parenchymal renal diseases. The emergence of this drug family has resulted from the recognition that CDKs and GSK-3s play critical roles in the progression and regression of many kidney diseases. CDK/GSK-3 inhibitors suppress pathogenic proliferation, apoptosis, and inflammation, and promote regeneration of injured tissue. Preclinical efficacy has now been demonstrated in mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, collapsing glomerulopathy, proliferative lupus nephritis, polycystic kidney diseases, diabetic nephropathy, and several forms of acute kidney injury. Novel biomarkers of therapy are aiding the process of drug development. This review will highlight these advancements in renal therapeutics.

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

7-Bromoindirubin-3'-oxime induces caspase-independent cell death.

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Synthesis and anticancer activity of new pyrrolocarbazoles and pyrrolo-beta-carbolines.

'Bended' 1, 3 or 'linear' 2 pyrrolidino-fused (aza)carbazoles were prepared and screened towards a few cancer-related targets. Whereas 'bended' derivatives 1 and 3 proved to be weakly toxic, several members of the 'linear' family strongly interact with DNA, especially derivative 28a.

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.