New forms of induction of apoptosis in aggressive lymphoma using peptides that interrupt the RAS / RAF interaction

Interpretation: RAS-RAF-MEK-ERK is a key pathway for apoptosis regulation in cancer cells. B-Raf-inhibitors such as PLX4032 peptide was developed by Institute Curie-Université Pierre et Marie Curie in order to induce apoptosis in cancer cells. Objectives: To demonstrate pro-apoptotic properties and survival outcome of EP2014/064243 peptide in murine aggressive lymphoma. Material and Methodology: BALBc mice with T-lymphoma were randomized assigned either in Group A (peptide+cyclophosphamide-CFM); Group B (peptides), Group C (CFM-control) or Control D (Cl-Na 0.9%-SF control group). Survival probability was calculated by Kaplan-Meier analysis. Apoptosis was detected using TUNEL technique. The protocol was approved by the Institutional Committee for Animal Care (CICUAL: T04-01-2015) Results: The median survival was 24 days (21.6-26.4) for placebo, 33 days (28.0-35.4) for the CFM monotherapy group, 33 (27.1-35.8) for the peptide group and 34 days (24,4-40) for CFM-peptide combined treatment (p<0.05). In lymph node tissue the mean TUNEL positive cells per field for each treatment group was 2, 12 and 13 and 35 for SF, CFM, peptide and combined therapy (p<0.05). Conclusion: These findings suggest that in murine aggressive lymphoma treated by an experimental peptide in addition with CFM, had an exponentially pro-apoptotic effect than CFM alone, suggesting that the peptide potentiated the anti-tumoural effect of CFM.

Correction to: T cell leukemia control via Ras-Raf pathway inhibition with peptides.

[This corrects the article on p. 172 in vol. 10, PMID: 29075346.].

T cell leukemia control via Ras-Raf pathway inhibition with peptides.

RATIONALE: RAS-RAF-MEK-ERK pathway has been considered a promising target for anticancer therapy. However, tumor cells may develop resistance against such drugs via hyperactivation of N-Ras, which explains why novel therapeut-ic approaches. In this sense, the Institute Curie- Université Pierre et Marie Curie (Paris 6) designed peptides in order to disturb Ras/Raf interaction which showed pro-apoptotic properties. These peptides were patented as WO2015001045 A2 (PCT/EP2014/064243)5. OBJECTIVE: In order to check the anti-tumoral action of WO2015001045 A2 peptides in a very aggressive BALB/c mice spontaneous leukemia called LB, we performed the present study. METHOD & RESULTS: 50 BALB/c mice inoculated with 106 LB tumor cells were randomly assigned either to control (placebo) or treatment group (that daily received 3 mg of peptide per kg of mice) during 30 days. By day 15 only 24% of the control group was alive vs. 100% of the treatment group. The average survival in treated group was 20,27 days while in control group the mean survival was 15,48 days. Either bone marrow, spleen or axillary nodes demonstrated a higher level of malignant T cell presence compare with treated group (89,78% ; 95,64% & 77,68% versus 72,45%, 80,23% & 63.44% respectively for each organ inspected. DISCUSSION: Our study demonstrated an improvement in survival curves in mice model affected by spontaneous T lymphoid leukemia when peptides WO2015001045 A2 were used. These peptides might be a valid option to become part of the therapeutic armory for malignant lymphoproliferative diseases control.

Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation.

P53 controls the growth and survival of cells by acting in response to a multitude of cellular stresses. It is, however, not yet fully understood how different p53 activation pathways result in either cell cycle arrest or apoptosis. We and others have described an N-terminally truncated p53 protein (p53/47) originating from a second translation initiation site in the p53 messenger RNA (mRNA), which can interact with p53 and impose altered stability and transactivation properties to p53 complexes. Here we show that cap-dependent and cap-independent mechanisms of initiation govern the translation of the p53 mRNA. Changes in synthesis of full-length p53 or p53/47 are regulated through distinct cell stress-induced pathways acting through separate regions of the p53 mRNA. We also show that some cytotoxic drugs require the presence of full-length p53 to induce apoptosis, whereas for others p53/47 is sufficient. This indicates that by harbouring alternative translation initiation sites, the p53 mRNA gives rise to different levels of the p53 isoforms which help to orchestrate the cell biological outcome of p53 activation in response to different types of cell stress. This sheds new light into the way p53 can integrate and differentiate a large multiplicity of changes in the cellular environment.

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations.

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.

SIAH-1 interacts with alpha-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis.

SIAH-1, a human homologue of the Drosophila seven in absentia (Sina), has been implicated in ubiquitin-mediated proteolysis of different target proteins through its N-terminal RING finger domain. SIAH-1 is also induced during p53-mediated apoptosis. Furthermore, SIAH-1-transfected breast cancer cell line MCF-7 exhibits an altered mitotic process resulting in multinucleated giant cells. Now, using the two-hybrid system, we identified two new SIAH interacting proteins: Kid (kinesin like DNA binding protein) and alpha-tubulin. We demonstrate that SIAH is involved in the degradation of Kid via the ubiquitin-proteasome pathway. Our results suggest that SIAH-1 but not its N-terminal deletion mutant, affects the mitosis by an enhanced reduction of kinesin levels. Our results imply, for the first time, SIAH-1 in regulating the degradation of proteins directly implicated in the mitotic process.

SIAH-1 inhibits cell growth by altering the mitotic process.

SIAH-1, the human homologue of the drosophila seven in absentia gene, is a p53-p21Waf-1 inducible gene. We report that stable transfection with SIAH-1 of the epithelial breast cancer cell line MCF-7 blocks its growth process. The transfectants show a redistribution of SIAH-1 protein within the nucleus, more specifically to the nuclear matrix, associated to dramatic changes in cell morphology and defective mitosis. Multinucleated giant cells (2-12 nuclei in more than 50% cells) were a most striking observation associated with tubulin spindle disorganization and defective cytokinesis. There were also present at high frequency abortive mitotic figures, DNA bridges and persistance of intercellular bridges and midbodies, along with an increased expression of p21Waf-1. These results indicate that the mechanism of growth arrest induced by SIAH-1 in MCF-7 cells involves disorganization of the mitotic program, mainly during nuclei separation and cytokinesis.

Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells.

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.

p21WAF-1 reorganizes the nucleus in tumor suppression.

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21WAF-1, by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21(WAF-1) drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and fluorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.

Isolation of 10 differentially expressed cDNAs in p53-induced apoptosis: activation of the vertebrate homologue of the drosophila seven in absentia gene.

We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature-sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death.