Transforming acidic coiled-coil 3 and Aurora-A interact in human thyrocytes and their expression is deregulated in thyroid cancer tissues

Aurora-A kinase has recently been shown to be deregulated in thyroid cancer cells and tissues. Among the Aurora-A substrates identified, transforming acidic coiled-coil (TACC3), a member of the TACC family, plays an important role in cell cycle progression and alterations of its expression occur in different cancer tissues. In this study, we demonstrated the expression of the TACC3 gene in normal human thyroid cells (HTU5), and its modulation at both mRNA and protein levels during cell cycle. Its expression was found, with respect to HTU5 cells, unchanged in cells derived from a benign thyroid follicular tumor (HTU42), and significantly reduced in cell lines derived from follicular (FTC-133), papillary (B-CPAP), and anaplastic thyroid carcinomas (CAL-62 and 8305C). Moreover, in 16 differentiated thyroid cancer tissues, TACC3 mRNA levels were found, with respect to normal matched tissues, reduced by twofold in 56% of cases and increased by twofold in 44% of cases. In the same tissues, a correlation between the expression of the TACC3 and Aurora-A mRNAs was observed. TACC3 and Aurora-A interact in vivo in thyroid cells and both proteins localized onto the mitotic structure of thyroid cells. Finally, TACC3 localization on spindle microtubule was no more observed following the inhibition of Aurora kinase activity by VX-680. We propose that Aurora-A and TACC3 interaction is important to control the mitotic spindle organization required for proper chromosome segregation

Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells

Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference

In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer

Molecular Requirements For Targeting The Polyamine Transport System. Synthesis And Biological Evaluation Of Polyamine-Anthracene Conjugates

A series of nine N1-(9-anthracenylmethyl)tetraamines (e.g., Ant-4,4,4-tetraamine) were synthesized and evaluated for cytotoxicity in L1210, α-difluoromethylornithine (DFMO)-treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. Surprisingly, the 3,3,4- and 3,4,3-tetraamine motifs had the same or decreased cytotoxicity in DFMO-treated L1210 cells, whereas the rest of the tetraamine systems were usually more cytotoxic and gave lower IC50 values in this treated cell line. The most sensitive derivatives to DFMO treatment were the Ant-4,4,3- and Ant-4,4,4-tetraamine analogues, which were 7 and 5 times more cytotoxic in DFMO-treated L1210 cells, respectively. Ki values for each of the anthracenylmethyl(Ant)-polyamine conjugates were determined in L1210 cells and revealed that these systems are high-affinity ligands for the polyamine transporter (PAT). Mixed results were observed in the CHO and CHO-MG assays. The 4,4,4- and 5,4,4-tetraamine motifs were 3 times more toxic to CHO cells with active polyamine transporters. For example, the Ant-4,4,4-tetraamine conjugate displayed IC50 values of 11 μM in CHO cells and 33 μM in CHO-MG cells, a PAT-deficient cell line. This suggested that these derivatives used the PAT in part to access cells. However, most of the other tetraamine derivatives had similar potencies in both the CHO and CHO-MG cell lines. In terms of vector design, higher affinity for the PAT (lower Ki values) did not translate into higher potency for the tetraamine conjugate. In contrast, the related triamine systems, which had micromolar Ki values in L1210 cells, were more efficacious and selective. In one case, the 4,4-triamine motif imparted 150-fold higher potency in CHO cells than the CHO-MG mutant. A deconvolution microscopy study in A375 melanoma cells revealed a rapid internalization of the Ant-4,4-triamine as fluorescent vesicles, whereas the Ant-4,4,4-tetraamine remained mostly at the cell surface. These findings help define the key characteristics required for selective delivery of polyamine-drug conjugates into cell types with active polyamine transporters

Synthesis And Biological Evaluation Of Dihydromotuporamine Derivatives In Cells Containing Active Polyamine Transporters

Dihydromotuporamine C (4) and its 4,4-triamine analogue (5) were synthesized in good yield using ring-closing metathesis (RCM) methods. Comparison of their biological activities (Ki determinations in L1210 cells and IC50 determinations in L1210, CHO, and CHO-MG cells) revealed that the motuporamine derivatives do not use the polyamine transporter (PAT) for cellular entry. Bioevaluation of a N1-(anthracen-9- ylmethyl)-n1-(ethyl)homospermidine control (7) revealed that the presence of a N1 tertiary amine center imparted a significant reduction in the PAT affinity of the polyamine conjugate and abolished its PAT-targeting selectivity. © 2005 American Chemical Society

Synthesis And Biological Evaluation Of N\u3csup\u3e1\u3c/sup\u3e-(Anthracen-9-Ylmethyl)Triamines As Molecular Recognition Elements For The Polyamine Transporter

An efficient modular synthesis of N1-substituted triamines containing different tether lengths between nitrogen centers was developed. A series of N1-(9-anthracenylmethyl)triamines were evaluated for biological activity in L1210 (murine leukemia), α-difluoromethylornithine (DFMO)treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. All triamines 8 had increased potency in DFMO-treated L1210 cells. The 4,4- and 5,4-triamine systems had the highest affinity for the polyamine transporter (PAT) with L1210 Ki values of 1.8 and 1.7 μM, respectively. This trend was also reflected in the CHO studies. Surprisingly, the respective 4,4- and 5,4-triamine systems had 150-fold and 38-fold higher cytotoxicity in CHO cells containing active polyamine transporters. Initial microscopy studies revealed the rapid formation of vesicular structures within A375 melanoma cells treated with the N1-(9- anthracenylmethyl)homospermidine (4,4-triamine) conjugate. In summary, the 4,4- and 5,4-triamines were identified as selective vector motifs to ferry anthracene into cells via the PAT

Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis in Drosophila S2 cells.

International audienceThe dynactin complex cooperates with the dynein complex in various systems for mitotic completion. Here we analysed the mitotic phenotype of Drosophila S2 cells following the knockdown of the dynactin subunit p150(Glued). We found that p150(Glued)-depleted cells were delayed in metaphase and that the centrosomes were poorly connected to mitotic spindle poles. In addition, anaphase occurred with asynchronous chromosome segregation. Although cyclin B was degraded in these anaphase cells, Aurora B, MEI-S322 and BubR1 were not released from the non-segregating chromosomes. We also found that the density and organisation of the central spindle were compromised, with Aurora B and polo kinases absent from the diminished number of microtubules. Pavarotti-KLP, a component of the centralspindlin complex required for the formation of stable microtubule bundles, was not immediately targeted to the plus ends of the microtubules following anaphase onset as happened in controls. Instead, it accumulated transiently at the cell cortex during early anaphase and its targeting to the central spindle was delayed. These data suggest that the dynactin complex contributes to cytokinesis by promoting stable targeting of the centralspindlin complex to microtubule plus ends at anaphase onset. The contribution of the dynein-dynactin complex to synchronous chromosome segregation and cytokinesis is discussed

Defining The Molecular Requirements For The Selective Delivery Of Polyamine Conjugates Into Cells Containing Active Polyamine Transporters

Several N1-substituted polyamines containing various spacer units between nitrogen centers were synthesized as their respective HCl salts. The N1-substituents included benzyl, naphthalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-ylmethyl. The polyamine spacer units ranged from generic (4,4-triamine, 4,3-triamine, and diaminooctane) spacers to more exotic [2-(ethoxy)ethanoxy-containing diamine, hydroxylated 4,3-triamine, and cyclohexylene-containing triamine] spacers. Two control compounds were also evaluated: N-(anthracen-9-ylmethyl)-butylamine and N-(anthracen-9-ylmethyl)-butanediamine. Biological activities in L1210 (murine leukemia), α-difluoromethylornithine (DFMO)-treated L1210, and Chinese hamster ovary (CHO) and its polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC50 cytotoxicity determinations. K i values for spermidine uptake were also determined in L1210 cells. Of the series studied, the N1-benzyl-4,4-triamine system 6 had significantly higher IC50 values (lower cytotoxicity) in the L1210, CHO, and CHO-MG cell lines. A cellular debenzylation process was observed in L1210 cells with 6 and generated free homospermidine. The size of the N1-arylmethyl substituent had direct bearing on the observed cytotoxicity in CHO-MG cells. The N1-naphthalenylmethyl, N′-anthracenylmethyl, and N′-pyrenylmethyl 4,4-triamines had similar toxicity (IC50s: ∼0.5 μM) in CHO cells, which have an active polyamine transporter (PAT). However, this series had IC50 values of \u3e 100 μM, 66.7 μM, and 15.5 μM, respectively, in CHO-MG cells, which are PAT-deficient. The observed lower cytotoxicity in the PAT-deficient CHO-MG cell line supported the premise that the conjugates use PAT for cellular entry. In general, moderate affinities for the polyamine transporter were observed for the N-arylmethyl 4,4-triamine series with their L1210 Ki values all near 3 μM. In summary, the 4,4-triamine motif was shown to facilitate entry of polyamine conjugates into cells containing active polyamine transporters

Development of Targeted Microbubbles for Ultrasound Molecular Imaging of Breast Cancer

National audienceA subtype of metastatic breast cancer has recently been identified, in which netrin-1 is over-expressed. Netrin-1 binds to dependence receptors inducing survival, while its absence actively triggers apoptosis. An anti-netrin-1 therapy is under development , which disrupts ligand-receptor interaction and induces apoptosis. To identify potential responders for such a targeted therapy, netrin-1 expression has to be analyzed. Molecular imaging with ultrasound (US) is able to detect the expression of a specific protein when using targeted contrast agents. Thus, we aim to develop an US molecular imaging approach using microbubbles (MB) functionalized with an anti-netrin-1 antibody. Binding was tested on purified netrin-1 protein in static and dynamic conditions. Anti-netrin-1 MB were validated in in-vitro assays and will be further tested in pre-clinical studies using metastatic breast cancer animal models

A Drosophila Model To Identify Polyamine-Drug Conjugates That Target The Polyamine Transporter In An Intact Epithelium

Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine - drug conjugates could be targeted to cancer cells via the polyamine transporter (PAT). We have previously reported the use of Chinese hamster ovary (CHO) cells and its PAT-deficient mutant cell line, CHO-MG, to screen anthracene - polyamine conjugates for their PAT-selective targeting ability. We report here a novel Drosophilabased model for screening anthracene-polyamine conjugates in a developing and intact epithelium (Drosophila imaginal discs), wherein cell-cell adhesion properties are maintained. Data from the Drosophila assay are consistent with previous results in CHO cells, indicating that the Drosophila epithelium has a PAT with vertebrate-like characteristics. This assay will be of use to medicinal chemists interested in screening drugs that use PAT for cellular entry, and it offers the possibility of genetic dissection of the polyamine transport process, including identification of a Drosophila PAT. © 2008 American Chemical Society