Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Development of A Flow Cytometry Assay for Measurement of CD4+ T Cell Proliferation

Thesis (Master's)--University of Washington, 2016-06

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

TRIB2 in human AML: a biological and clinical investigation

Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest of cells at a very early stage of myeloid cell differentiation. The biological and clinical heterogeneity of this disease complicates treatment and highlights the significance of understanding the underlying causes of AML, which may constitute potential therapeutic targets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potent murine oncogene capable of inducing transplantable AML with complete penetrance. The pathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of the full length isoform of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα p42). The role of TRIB2 in human AML cells, however, has not been systematically investigated or targeted. Across human cancers, TRIB2 oncogenic activity was found to be associated with its elevated expression. In the context of AML, TRIB2 overexpression was suggested to be associated with the large and heterogeneous subset of cytogenetically normal AML patients. Based upon the observation that overexpression of TRIB2 has a role in cellular transformation, the effect of modulating its expression in human AML was examined in a human AML cell line that expresses high levels of TRIB2, U937 cells. Specific suppression of TRIB2 led to impaired cell growth, as a consequence of both an increase in apoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2 silencing strongly reduced progression of the U937 in vivo xenografts, accompanied by detection of a lower spleen weight when compared with mice transplanted with TRIB2- expressing control cells. Gene expression analysis suggested that TRIB2 modulates apoptosis and cell-cycle sensitivity by influencing the expression of a subset of genes known to have implications on these phenotypes. Furthermore, TRIB2 was found to be expressed in a significant subset of AML patient samples analysed. To investigate whether increased expression of this gene could be afforded prognostic significance, primary AML cells with dichotomized levels of TRIB2 transcripts were evaluated in terms of their xenoengraftment potential, an assay reported to correlate with disease aggressiveness observed in humans. A small cohort of analysed samples with higher TRIB2 expression did not associate with preferential leukaemic cell engraftment in highly immune-deficient mice, hence, not predicting for an adverse prognosis. However, further experiments including a larger cohort of well characterized AML patients would be needed to clarify TRIB2 significance in the diagnostic setting. Collectively, these data support a functional role for TRIB2 in the maintenance of the oncogenic properties of human AML cells and suggest TRIB2 can be considered a rational therapeutic target. Proteasome inhibition has emerged as an attractive target for the development of novel anti-cancer therapies and results from translational research and clinical trials support the idea that proteasome inhibitors should be considered in the treatment of AML. The present study argued that proteasome inhibition would effectively inhibit the function of TRIB2 by abrogating C/EBPα p42 protein degradation and that it would be an effective pharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cell models expressing high levels of TRIB2 were successfully targeted by treatment with proteasome inhibitors, as demonstrated by multiple measurements that included increased cytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo. Mechanistically, it was shown that block of the TRIB2 degradative function led to an increase of C/EBPα p42 and that response was specific to the TRIB2-C/EBPα axis. Specificity was addressed by a panel of experiments showing that U937 cells (express detectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitor bortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression and that ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negative leukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased following TRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPα axis. Together these findings provide pre-clinical evidence that TRIB2- expressing AML cells can be pharmacologically targeted with proteasome inhibition due, in part, to blockage of the TRIB2 proteolytic function on C/EBPα p42. A large body of evidence indicates that AML arises through the stepwise acquisition of genetic and epigenetic changes. Mass spectrometry data has identified an interaction between TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5 (PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effective targeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5 cooperation was investigated in order to gain a deeper understanding of the molecular network in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data set was interrogated for PRMT5 expression levels and the primary enzyme responsible for symmetric dimethylation was found to be transcribed at significantly higher levels in AML patients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cell line was shown to reduce the transformative phenotype in the high expressing TRIB2 AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain the leukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interacting protein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlying mechanism and the functional significance of this interplay. In summary, the present study provides experimental evidence that TRIB2 has an important oncogenic role in human AML maintenance and, importantly in such a molecularly heterogeneous disease, provides the rational basis to consider proteasome inhibition as an effective targeting strategy for AML patients with high TRIB2 expression. Finally, the identification of PRMT5 as a TRIB2-interacting protein opens a new level of regulation to consider in AML. This work may contribute to our further understanding and therapeutic strategies in acute leukaemias.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

Estudo da função da proteína Mob1 na estrutura dos centrossomas

Dissertação de Mestrado, Oncobiologia - Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Inhibition of proliferation, migration and invasion of human non-small cell lung cancer cell line A549 by phlomisoside F from Phlomis younghusbandii Mukerjee

Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting. Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF. Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.

Recent advances in cancer therapy targeting proteins involved in DNA double-strand break repair

Damage to genetic material represents a persistent and ubiquitous threat to genomic stability. Once DNA damage is detected, a multifaceted signaling network is activated that halts the cell cycle, initiates repair, and in some instances induces apoptotic cell death. In this article, we will review DNA damage surveillance networks, which maintain the stability of our genome, and discuss the efforts underway to identify chemotherapeutic compounds targeting the core components of DNA double-strand breaks (DSB) response pathway. The majority of tumor cells have defects in maintaining genomic stability owing to the loss of an appropriate response to DNA damage. New anticancer agents are exploiting this vulnerability of cancer cells to enhance therapeutic indexes, with limited normal tissue toxicity. Recently inhibitors of the checkpoint kinases Chk1 and Chk2 have been shown to sensitize tumor cells to DNA damaging agents. In addition, the treatment of BRCA1- or BRCA2-deficient tumor cells with poly(ADP-ribose) polymerase (PARP) inhibitors also leads to specific tumor killing. Due to the numerous roles of p53 in genomic stability and its defects in many human cancers, therapeutic agents that restore p53 activity in tumors are the subject of multiple clinical trials. In this article we highlight the proteins mentioned above and catalog several additional players in the DNA damage response pathway, including ATM, DNA-PK, and the MRN complex, which might be amenable to pharmacological interventions and lead to new approaches to sensitize cancer cells to radio- and chemotherapy. The challenge is how to identify those patients most receptive to these treatments.