TCTN2:a novel tumor marker with oncogenic properties

Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.</p

Identification of New Hematopoietic Cell Subsets with a Polyclonal Antibody Library Specific for Neglected Proteins

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs

Mutant Prourokinase with Adjunctive C1-Inhibitor Is an Effective and Safer Alternative to tPA in Rat Stroke

A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/− C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings

Schematic representation of the antisera library generation.

<p>Schematic representation of the antisera library generation.</p

Results of sera screening by FACS on PBL and Cord Blood cells.

<p>(A) FACS analysis of sera positive on PBLs. PBLs were stained with the indicated sera at the optimal dilution point (1∶50 to 1∶200). The samples were stained also with anti CD3, anti CD19 and anti CD56 mAbs to analyze the sera reactivity upon gating on the different subpopulations. A plot representative of five different donors is shown for each serum. (B) KRTCAP-3 specific serum recognizes PHA-treated cells. PBMCs are treated for 24 hours with 1 µg/ml of PHA. After the treatment both un-stimulated and treated cells are stained with the KRTCAP-3-specific serum. (C) FACS analysis of sera positive on cord blood cells. Cord blood mononuclear cells are stained with the indicated sera at the optimal concentration (1∶50 to 1∶100). The samples are stained also with anti CD45 and anti CD34 mAbs to perform the analysis upon gating on CD34highCD45dim. A plot representative of a least 3 independent donors is shown. Il all the cases (A,B,C,) a staining with the serum of not immunized mice was used as negative control. (D) RT-PCR analysis. a- cDNA from total PBMC were amplified with primers specific for the indicated proteins. b- cDNA from both un-stimulated and PHA-treated PBMC was amplified with KRTCAP-3 specific primers. KRTCAP3 expression is up regulated two to three times. Beta actin amplification is used as normalization. c- cDNA samples from CD34+CD45dim cells were generated by retro-transcription of RNA extracted from a pool of CD34 positive cells from 2–3 independent cord blood units magnetically purified using the Miltenyi CD34 microbeads kit according the manufacturer instruction. The purity of the CD34+CD45dim cells was usually >99%. The samples were amplified with primers specific for the indicated proteins and described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034395#s2" target="_blank">Methods</a> section.</p

Assessment of antisera specificity on Hela transfected cells.

<p>Hela cells were transiently transfected with a myc-tag version of the proteins identified with the sera library. At 24 hours from the transfection cells were lysated as described in the Method section. 40 µg of total proteins were loaded on SDS page and a WB analysis was performed using both an anti myc mAb (9E10 clone) and the corresponding antiserum. (A) WB analysis of Hela cells transfected with CRISP-1 and MOSC-1. In both the cases the anti myc mAb and the specific antiserum recognized a protein of the expected molecular weight that is not present in the cells transfected with the mock vector. A comparable result was obtained wit KRTCAP-3 (B), TMCC-1 (C), TMEM38B (D) and SUSD3 (E) transfected cells. The WB analysis of GSG1-L cells (E) and LPPR2 cells (F) shows that neither the anti myc nor the specific antiserum is able to recognize in a specific way a protein in transfected cells.</p

Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.

<p>Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.</p