Caspase-generated fragment of the Met receptor favors apoptosis via the intrinsic pathway independently of its tyrosine kinase activity

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas the deregulation of Met signaling is associated with tumorigenesis. While ligand-activated Met promotes survival, caspase-dependent generation of the p40 Met fragment leads to apoptosis induction – hallmark of the dependence receptor. Although the survival signaling pathways induced by Met are well described, the pro-apoptotic signaling pathways are unknown. We show that, although p40 Met contains the entire kinase domain, it accelerates apoptosis independently of kinase activity. In cell cultures undergoing apoptosis, the fragment shows a mitochondrial localization, required for p40 Met-induced cell death. Fulminant hepatic failure induced in mice leads to the generation of p40 Met localized also in the mitochondria, demonstrating caspase cleavage of Met in vivo. According to its localization, the fragment induces mitochondrial permeabilization, which is inhibited by Bak silencing and Bcl-xL overexpression. Moreover, Met silencing delays mitochondrial permeabilization induced by an apoptotic treatment. Thus, the Met-dependence receptor in addition to its well-known role in survival signaling mediated by its kinase activity, also participates in the intrinsic apoptosis pathway through the generation of p40 Met – a caspase-dependent fragment of Met implicated in the mitochondrial permeabilization process

Cancer - Cell survival guide

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62618/1/431035a.pd

A Novel Motif Identified in Dependence Receptors

Programmed cell death signaling is a critical feature of development, cellular turnover, oncogenesis, and neurodegeneration, among other processes. Such signaling may be transduced via specific receptors, either following ligand binding—to death receptors—or following the withdrawal of trophic ligands—from dependence receptors. Although dependence receptors display functional similarities, no common structural domains have been identified. Therefore, we employed the Multiple Expectation Maximization for Motif Elicitation and the Motif Alignment and Search Tool software programs to identify a novel transmembrane motif, dubbed dependence-associated receptor transmembrane (DART) motif, that is common to all described dependence receptors. Of 3,465 human transmembrane proteins, 25 (0.7%) display the DART motif. The predicted secondary structure features an alpha helical structure, with an unusually high percentage of valine residues. At least four of the proteins undergo regulated intramembrane proteolysis. To date, we have not identified a function for this putative domain. We speculate that the DART motif may be involved in protein processing, interaction with other proteins or lipids, or homomultimerization

Down-Regulation of Neogenin Accelerated Glioma Progression through Promoter Methylation and Its Overexpression in SHG-44 Induced Apoptosis

Dependence receptors have been proved to act as tumor suppressors in tumorigenesis. Neogenin, a DCC homologue, well known for its fundamental role in axon guidance and cellular differentiation, is also a dependence receptor functioning to control apoptosis. However, loss of neogenin has been reported in several kinds of cancers, but its role in glioma remains to be further investigated.Western blot analysis showed that neogenin level was lower in glioma tissues than in their matching surrounding non-neoplastic tissues (n = 13, p<0.01). By immunohistochemical analysis of 69 primary and 16 paired initial and recurrent glioma sections, we found that the loss of neogenin did not only correlate negatively with glioma malignancy (n = 69, p<0.01), but also glioma recurrence (n = 16, p<0.05). Kaplan-Meier plot and Cox proportional hazards modelling showed that over-expressive neogenin could prolong the tumor latency (n = 69, p<0.001, 1187.6 ± 162.6 days versus 687.4 ± 254.2 days) and restrain high-grade glioma development (n = 69, p<0.01, HR: 0.264, 95% CI: 0.102 to 0.687). By Methylation specific polymerase chain reaction (MSP), we reported that neogenin promoter was methylated in 31.0% (9/29) gliomas, but absent in 3 kinds of glioma cell lines. Interestingly, the prevalence of methylation in high-grade gliomas was higher than low-grade gliomas and non-neoplastic brain tissues (n = 33, p<0.05) and overall methylation rate increased as glioma malignancy advanced. Furthermore, when cells were over-expressed by neogenin, the apoptotic rate in SHG-44 was increased to 39.7% compared with 8.1% in the blank control (p<0.01) and 9.3% in the negative control (p<0.01).These observations recapitulated the proposed role of neogenin as a tumor suppressor in gliomas and we suggest its down-regulation owing to promoter methylation is a selective advantage for glioma genesis, progression and recurrence. Furthermore, the induction of apoptosis in SHG-44 cells after overexpression of neogenin, indicated that neogenin could be a novel target for glioma therapy

Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction

Nephrotic syndrome (NS) is characterized by structural changes in the actin‐rich foot processes of glomerular podocytes. We previously identified high concentrations of the small heat shock protein hsp27 within podocytes as well as increased glomerular accumulation and phosphorylation of hsp27 in puromycin aminonucleoside (PAN) ‐induced experimental NS. Here we analyzed murine podocytes stably transfected with hsp27 sense, antisense, and vector control constructs using a newly developed in vitro PAN model system. Cell morphology and the microfilament structure of untreated sense and antisense transfectants were altered compared with controls. Vector cell survival, polymerized actin content, cell area, and hsp27 content increased after 1.25 μg/ml PAN treatment and decreased after 5.0 μg/ml treatment. In contrast, sense cells were unaffected by 1.25 μg/ml PAN treatment whereas antisense cells showed decreases or no changes in all parameters. Treatment of sense cells with 5.0 μ g/ml PAN resulted in increased cell survival and cell area whereas antisense cells underwent significant decreases in all parameters. Hsp27 provided dramatic protection against PAN‐induced microfilament disruption in sense > vector > antisense cells. We conclude that hsp27 is able to regulate both the morphological and actin cytoskeletal response of podocytes in an in vitro model of podocyte injury.—Smoyer, W. E., Ransom, R. F. Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction. FASEB J. 16, 315–326 (2002)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154256/1/fsb2fj010681com.pd

A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A.

BACKGROUND: Multiple endocrine neoplasias type 2A (MEN 2A) is a dominantly inherited cancer syndrome. Missence mutations in the codon encoding cysteine 634 of the ret proto-oncogene have been found in 85% of the MEN 2A families. The main tumour type always present in MEN 2A is medullar thyroid carcinoma (MTC). Only 25% of all MTC are hereditary, and generally they are identified by a careful family history. However, some familial MTCs are not easily detected by this means and underdiagnosis of MEN 2A is suspected. METHODS: DNA samples from MEN 2A patients were amplified by PCR. The products were incubated with the restriction enzyme Bst ApI or Bgl I. The samples were loaded in non-denaturing 10% Polyacrilamyde Gel and run at 120 volts for 40 min. The gels were stained with 10 μg/ml ethidium bromide, and the bands were visualized under a UV lamp. RESULTS: We developed a PCR-mutagenic method to check the integrity of the three bases of the cysteine 634 codon. CONCLUSION: The method can be used to detect inherited mutations in MTC patients without a clear family history. The method is relatively simple to use as a routine test in these patients to decrease the underdiagnosis of MEN 2A. In addition, the assay can be used to screen affected families with any mutation in cysteine 634

Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1s function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells

RET signalling provides tumorigenic mechanism and tissue specificity for AIP-related somatotrophinomas

International audienceIt is unclear how loss-of-function germline mutations in the widely-expressed co-chaperone AIP , result in young-onset growth hormone secreting pituitary tumours. The RET receptor, uniquely co-expressed in somatotrophs with PIT1, induces apoptosis when unliganded, while RET supports cell survival when it is bound to its ligand. We demonstrate that at the plasma membrane, AIP is required to form a complex with monomeric-intracellular-RET, caspase-3 and PKCδ resulting in PIT1/CDKN2A-ARF/p53-apoptosis pathway activation. AIP-deficiency blocks RET/caspase-3/PKCδ activation preventing PIT1 accumulation and apoptosis. The presence or lack of the inhibitory effect on RET-induced apoptosis separated pathogenic AIP variants from non-pathogenic ones. We used virogenomics in neonatal rats to demonstrate the effect of mutant AIP protein on the RET apoptotic pathway in vivo. In adult male rats altered AIP induces elevated IGF-1 and gigantism, with pituitary hyperplasia through blocking the RET-apoptotic pathway. In females, pituitary hyperplasia is induced but IGF-1 rise and gigantism are blunted by puberty. Somatotroph adenomas from pituitary-specific Aip -knockout mice overexpress the RET-ligand GDNF, therefore, upregulating the survival pathway. Somatotroph adenomas from patients with or without AIP mutation abundantly express GDNF, but AIP -mutated tissues have less CDKN2A-ARF expression. Our findings explain the tissue-specific mechanism of AIP-induced somatotrophinomas and provide a previously unknown tumorigenic mechanism, opening treatment avenues for AIP -related tumours

miRNA in situ hybridization in circulating tumor cells - MishCTC

irculating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.JJDM thanks the Spanish Ministerio de Economía y Competitividad for a Ramón y Cajal Fellowship (Grant CTQ2012-34778). This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Program (FP7-PEOPLE-2011-CIG-Project Number 294142 to RMSM and FP7-PEOPLE-2012-CIG-Project Number 322276 toJJDM). Thisresearch wasalso partially supported by Consejeria de Salud de la Junta de Andalucı ́ a (PI0294-2012)