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YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis., (© 2024. The Author(s).)

Cardiac alteration due to chronic kidney disease is described by tissue fibrosis. This remodeling involves myofibroblasts of various origins, including epithelial or endothelial to mesenchymal transitions. In addition, obesity and insulin resistance together or separately seem to exacerbate cardiovascular risk in chronic kidney disease (CKD). The main objective of this study was to assess if pre-existing metabolic disease exacerbates CKD-induced cardiac alterations. In addition, we hypothesised that endothelial to mesenchymal transition participates in this enhancement of cardiac fibrosis. Rats fed cafeteria type diet for 6 months underwent a subtotal nephrectomy at 4 months. Cardiac fibrosis was evaluated by histology and qRT-PCR. Collagens and macrophages were quantified by immunohistochemistry. Endothelial to mesenchymal transitions were assessed by qRT-PCR (CD31, VE-cadherin, α-SMA, nestin) and also by CD31 immunofluorescence staining. Rats fed with cafeteria type regimen were obese, hypertensive and insulin resistant. Cardiac fibrosis was predominant in CKD rats and was highly majored by cafeteria regimen. Collagen-1 and nestin expressions were higher in CKD rats, independently of regimen. Interestingly, in rats with CKD and cafeteria diet we found an increase of CD31 and α-SMA co-staining with suggest an implication of endothelial to mesenchymal transition during heart fibrosis. We showed that rats already obese and insulin resistant had an enhanced cardiac alteration to a subsequent renal injury. Cardiac fibrosis process could be supported by a involvement of the endothelial to mesenchymal transition phenomenon., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years, the utility and efficiency of automated procedures for cognitive assessment in psychology and neuroscience have been demonstrated in non-human primates (NHP). This approach mimics conventional shaping principles of breaking down a final desired behavior into smaller components that can be trained in a staircase manner. When combined with home-cage-based approaches, this could lead to a reduction in human workload, enhancement in data quality, and improvement in animal welfare. However, to our knowledge, there are no reported attempts to develop automated training and testing protocols for long-tailed macaques ( Macaca fascicularis ), a ubiquitous NHP model in neuroscience and pharmaceutical research. In the current work, we present the results from 6 long-tailed macaques that were trained using an automated unsupervised training (AUT) protocol for introducing the animals to the basics of a two-alternative choice (2 AC) task where they had to discriminate a conspecific vocalization from a pure tone relying on images presented on a touchscreen to report their response. We found that animals (1) consistently engaged with the device across several months; (2) interacted in bouts of high engagement; (3) alternated peacefully to interact with the device; and (4) smoothly ascended from step to step in the visually guided section of the procedure, in line with previous results from other NHPs. However, we also found (5) that animals' performance remained at chance level as soon as the acoustically guided steps were reached; and (6) that the engagement level decreased significantly with decreasing performance during the transition from visual to acoustic-guided sections. We conclude that with an autonomous approach, it is possible to train long-tailed macaques in their social group using computer vision techniques and without dietary restriction to solve a visually guided discrimination task but not an acoustically guided task. We provide suggestions on what future attempts could take into consideration to instruct acoustically guided discrimination tasks successfully., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cabrera-Moreno, Jeanson, Jeschke and Calapai.)

Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo., Competing Interests: Piona Dariavach and Pierre Martineau are inventors of the patent application No EP 22306229 “SYK INHIBITORS FOR USE IN THE TREATMENT OF CANCER”. All other authors confirm that they have no competing interests.

During early stages of type 2 diabetes, named prediabetes, pancreatic β-cells compensate for insulin resistance through increased insulin secretion in order to maintain normoglycemia. Obesity leads to the development of ectopic fat deposits, among which peri-pancreatic white adipose tissue (pWAT) can communicate with β-cells through soluble mediators. Thus we investigated whether pWAT produced oxygenated lipids, namely isoprostanes and neuroprostanes and whether they can influence β-cell function in obesity. In the Zucker fa/fa rat model, pWAT and epididymal white adipose tissue (eWAT) displayed different inflammatory profiles. In obese rats, pWAT, but not eWAT, released less amounts of 5-F 2t -isoprostanes, 15-F 2t -isoprostanes, 4-F 4t -neuroprostanes and 10-F 4t -neuroprostane compared to lean animals. These differences could be explained by a greater induction of antioxidant defenses enzymes such as SOD-1, SOD-2, and catalase in pWAT of obese animals compared to eWAT. In addition, sPLA2 IIA, involved in the release of isoprostanoids from cellular membranes, was decreased in pWAT of obese animals, but not in eWAT, and may also account for the reduced release of oxidized lipids by this tissue. At a functional level, 15-F 2t -isoprostane epimers, but not 5-F 2t -isoprostanes, were able to decrease glucose-induced insulin secretion in pancreatic islets from Wistar rats. This effect appeared to be mediated through activation of the thromboxane A2 receptor and reduction of cAMP signaling in pancreatic islets. In conclusion, through the removal of an inhibitory tone exerted by isoprostanes, we have shown, for the first time, a new mechanism allowing β-cells to compensate for insulin resistance in obesity that is linked to a biocommunication between adipose tissue and β-cells., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Excessive fat consumption leads to the development of ectopic adipose tissues, affecting the organs they surround. Peripancreatic adipose tissue is implicated in glucose homeostasis regulation and can be impaired in obesity. High palm oil consumption's effects on health are still debated. We hypothesised that crude and refined palm oil high-fat feeding may have contrasting effects on peripancreatic adipocyte hypertrophy, inflammation and lipid oxidation compound production in obese rats. In Wistar rats, morphological changes, inflammation and isoprostanoid production following oxidative stress were assessed in peripancreatic adipose tissue after 12 weeks of diets enriched in crude or refined palm oil or lard (56% energy from fat in each case) versus a standard chow diet (11% energy from fat). Epididymal white and periaortic brown adipose tissues were also included in the study. A refined palm oil diet disturbed glucose homeostasis and promoted lipid deposition in periaortic locations, as well as adipocyte hypertrophy, macrophage infiltration and isoprostanoid (5-F 2c -isoprostane and 7 (RS) -ST-Δ 8 -11-dihomo-isofuran) production in peripancreatic adipose tissue. Crude palm oil induced a lower impact on adipose deposits than its refined form and lard. Our results show that the antioxidant composition of crude palm oil may have a protective effect on ectopic adipose tissues under the condition of excessive fat intake.

In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) for 8weeks. After 4weeks, rats were infused or not with angiotensin II (200ng·kg -1 ·min -1 , sc) for 4weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hypertrophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling., (Copyright © 2017 Elsevier B.V. All rights reserved.)

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

The presentation of intense, low-frequency (LF) sound to the human ear can cause very slow, sinusoidal oscillations of cochlear sensitivity after LF sound offset, coined the "Bounce" phenomenon. Changes in level and frequency of spontaneous otoacoustic emissions (SOAEs) are a sensitive measure of the Bounce. Here, we investigated the effect of LF sound level and frequency on the Bounce. Specifically, the level of SOAEs was tracked for minutes before and after a 90-s LF sound exposure. Trials were carried out with several LF sound levels (93 to 108 dB SPL corresponding to 47 to 75 phons at a fixed frequency of 30 Hz) and different LF sound frequencies (30, 60, 120, 240 and 480 Hz at a fixed loudness level of 80 phons). At an LF sound frequency of 30 Hz, a minimal sound level of 102 dB SPL (64 phons) was sufficient to elicit a significant Bounce. In some subjects, however, 93 dB SPL (47 phons), the lowest level used, was sufficient to elicit the Bounce phenomenon and actual thresholds could have been even lower. Measurements with different LF sound frequencies showed a mild reduction of the Bounce phenomenon with increasing LF sound frequency. This indicates that the strength of the Bounce not only is a simple function of the spectral separation between SOAE and LF sound frequency but also depends on absolute LF sound frequency, possibly related to the magnitude of the AC component of the outer hair cell receptor potential., Competing Interests: Compliance with Ethical Standards All experiments were approved by the ethics committee of the University Hospital of the Ludwig-Maximilians-University Munich, Germany, in agreement with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans, and all subjects gave their written informed consent.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disease due to functional or ultra-structural defects of motile cilia. Affected individuals display recurrent respiratory-tract infections; most males are infertile as a result of sperm flagellar dysfunction. The great majority of the PCD-associated genes identified so far encode either components of dynein arms (DAs), which are multiprotein-ATPase complexes essential for ciliary motility, or proteins involved in DA assembly. To identify the molecular basis of a PCD phenotype characterized by central complex (CC) defects but normal DA structure, a phenotype found in ∼15% of cases, we performed whole-exome sequencing in a male individual with PCD and unexplained CC defects. This analysis, combined with whole-genome SNP genotyping, identified a homozygous mutation in DNAJB13 (c.833T>G), a gene encoding a HSP40 co-chaperone whose ortholog in the flagellated alga Chlamydomonas localizes to the radial spokes. In vitro studies showed that this missense substitution (p.Met278Arg), which involves a highly conserved residue of several HSP40 family members, leads to protein instability and triggers proteasomal degradation, a result confirmed by the absence of endogenous DNAJB13 in cilia and sperm from this individual. Subsequent DNAJB13 analyses identified another homozygous mutation in a second family; the study of DNAJB13 transcripts obtained from airway cells showed that this mutation (c.68+1G>C) results in a splicing defect consistent with a loss-of-function mutation. Overall, this study, which establishes mutations in DNAJB13 as a cause of PCD, unveils the key role played by DNAJB13 in the proper formation and function of ciliary and flagellar axonemes in humans., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans., (© 2016 WILEY PERIODICALS, INC.)

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive condition resulting from structural and/or functional defects of the axoneme in motile cilia and sperm flagella. The great majority of mutations identified so far involve genes whose defects result in dynein-arm anomalies. By contrast, PCD due to CC/RS defects (those in the central complex [CC] and radial spokes [RSs]), which might be difficult to diagnose, remains mostly unexplained. We identified non-ambiguous RSPH3 mutations in 5 of 48 independent families affected by CC/RS defects. RSPH3, whose ortholog in the flagellated alga Chlamydomonas reinhardtii encodes a RS-stalk protein, is mainly expressed in respiratory and testicular cells. Its protein product, which localizes within the cilia of respiratory epithelial cells, was undetectable in airway cells from an individual with RSPH3 mutations and in whom RSPH23 (a RS-neck protein) and RSPH1 and RSPH4A (RS-head proteins) were found to be still present within cilia. In the case of RSPH3 mutations, high-speed-videomicroscopy analyses revealed the coexistence of immotile cilia and motile cilia with movements of reduced amplitude. A striking feature of the ultrastructural phenotype associated with RSPH3 mutations is the near absence of detectable RSs in all cilia in combination with a variable proportion of cilia with CC defects. Overall, this study shows that RSPH3 mutations contribute to disease in more than 10% of PCD-affected individuals with CC/RS defects, thereby allowing an accurate diagnosis to be made in such cases. It also unveils the key role of RSPH3 in the proper building of RSs and the CC in humans., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive respiratory disorder resulting from defects of motile cilia. Various axonemal ultrastructural phenotypes have been observed, including one with so-called central-complex (CC) defects, whose molecular basis remains unexplained in most cases. To identify genes involved in this phenotype, whose diagnosis can be particularly difficult to establish, we combined homozygosity mapping and whole-exome sequencing in a consanguineous individual with CC defects. This identified a nonsense mutation in RSPH1, a gene whose ortholog in Chlamydomonas reinhardtii encodes a radial-spoke (RS)-head protein and is mainly expressed in respiratory and testis cells. Subsequent analyses of RSPH1 identified biallelic mutations in 10 of 48 independent families affected by CC defects. These mutations include splicing defects, as demonstrated by the study of RSPH1 transcripts obtained from airway cells of affected individuals. Wild-type RSPH1 localizes within cilia of airway cells, but we were unable to detect it in an individual with RSPH1 loss-of-function mutations. High-speed-videomicroscopy analyses revealed the coexistence of different ciliary beating patterns-cilia with a normal beat frequency but abnormal motion alongside immotile cilia or cilia with a slowed beat frequency-in each individual. This study shows that this gene is mutated in 20.8% of individuals with CC defects, whose diagnosis could now be improved by molecular screening. RSPH1 mutations thus appear as a major etiology for this PCD phenotype, which in fact includes RS defects, thereby unveiling the importance of RSPH1 in the proper building of CCs and RSs in humans., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: CCDC39 and CCDC40 genes have recently been implicated in primary ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal disorganisation; their contribution to the disease is, however, unknown. Aiming to delineate the CCDC39/CCDC40 mutation spectrum and associated phenotypes, this study screened a large cohort of patients with IDA defects, in whom clinical and ciliary phenotypes were accurately described., Methods: All CCDC39 and CCDC40 exons and intronic boundaries were sequenced in 43 patients from 40 unrelated families. The study recorded and compared clinical features (sex, origin, consanguinity, laterality defects, ages at first symptoms and at phenotype evaluation, neonatal respiratory distress, airway infections, nasal polyposis, otitis media, bronchiectasis, infertility), ciliary beat frequency, and quantitative ultrastructural analyses of cilia and sperm flagella., Results: Biallelic CCDC39 or CCDC40 mutations were identified in 30/34 (88.2%) unrelated families with IDA defects associated with axonemal disorganisation (22 and eight families, respectively). Fourteen of the 28 identified mutations are novel. No mutation was found in the six families with isolated IDA defects. Patients with identified mutations shared a similar phenotype, in terms of both clinical features and ciliary structure and function. The sperm flagellar ultrastructure, analysed in 4/7 infertile males, showed evidence of abnormalities similar to the ciliary ones., Conclusions: CCDC39 and CCDC40 mutations represent the major cause of PCD with IDA defects and axonemal disorganisation. Patients carrying CCDC39 or CCDC40 mutations are phenotypically indistinguishable. CCDC39 and CCDC40 analyses in selected patients ensure mutations are found with high probability, even if clinical or ciliary phenotypes cannot prioritise one analysis over the other.

Frequent deregulation of cyclin-dependent kinase (CDK) activation associated with loss of cell cycle control was found in most of human cancers. A recent development of a new class of antineoplasic agents targeting the cell cycle emerged as a small molecule CDK inhibitor, roscovitine, which presents potential antiproliferative and antitumoral effects in human tumors. Additional studies reported that roscovitine combined with cytotoxic agents can cooperate with DNA damage to activate p53 protein. However, little is known about the biological effect of roscovitine combined with ionizing radiation (IR) in human carcinoma, and no studies were reported thus far in p53 mutated carcinoma. In the breast cancer cell line MDA-MB 231, which lacks a functional p53 protein, we found a strong radiosensitization effect of roscovitine in vitro by clonogenic survival assay and in vivo in MDA-MB 231 xenograft model. Using Pulse Field Gel Electrophoresis, a strong impairment in DNA-double-strand break rejoining was observed after roscovitine and IR treatment as compared with IR alone. Cell cycle analysis showed a G(2) delay and no increase in radiation-induced apoptosis in the cells treated with IR or roscovitine and IR. On the other hand, we found a significant induction in micronuclei frequency after roscovitine and IR treatment as compared with IR alone. This effect was also observed in BALB murine cells in contrast to SCID murine cells, which are deficient in DNA-PKcs, suggesting a possible DNA-double-strand break repair defect in the nonhomologous end joining pathways. In MDA-MB 231 cells, the radiosensitization effect of roscovitine was associated with an inhibition of the DNA-dependent protein kinase activity caused by a marked decrease in Ku-DNA binding by using the electrophoretic mobility shift assay. In conclusion, we found a novel effect on DNA repair of the CDK inhibitor roscovitine, which acts as a radiosensitizer in vitro and in vivo in breast cancer cells lacking a functional p53.

To gain new insights regarding the role of Ku, the DNA-PK DNA-binding component, during lentiviral DNA integration, we have investigated the HIV-1 replication in Ku80-depleted human cells. CEM4fx cells underexpressing the Ku80 factor were selected after transduction by a retroviral vector expressing the Ku80 full-length antisense sequence. De novo infection experiment with NL4.3 HIV-1 strain led to the observation that the viral replication was delayed in the Ku80-depleted cells. Early events of the replicative cycle, including nuclear import of the viral DNA, were not affected. In contrast, the formation of the 2-LTR circles was impaired, thus demonstrating the implication of Ku in HIV-1 DNA circularization, for the first time in human cells. Furthermore, the detection of integrated proviruses by an Alu-LTR-nested PCR amplification method was affected in cells underexpressing Ku80. These results suggest that this factor may also be involved in the mechanisms leading to the stable establishment of HIV-1 provirus.

Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

Fourteen catechol and bis-catechol derivatives have been synthesised and tested for their HIV-1 inhibitory activities. The six more active molecules have been tested for their antiviral activity and cytotoxicity. We have found that bis-catechols 1 and 2 are the most active HIV-1 integrase inhibitor whereas the best antiviral compound is 4.