Cytotoxic lymphocytes; instigators of dramatic target cell death

Most mammalian cells are constantly threatened by viral infection and oncogenic transformation. To maintain healthy function of organs and tissues it is critical that afflicted cells are efficiently detected and removed. Cytotoxic lymphocytes (CL) are chiefly responsible for efficiently seeking out and eliminating damaged or infected cells. It is known that CLs must specifically recognize and bind to their targets, but the molecular events that occur within the target cell that lead to its death are still poorly understood. The two main processes initiated by CLs to induce target cell death are mediated by ligation of surface receptors or release of toxic proteins from secretory granules (granule exocytosis) of the CL. Here we review some of the key findings that have defined our knowledge of the granule exocytosis-mediated pathways to CL-mediated killing and discuss recent insights that challenge conventional views in the important area of CL effector function.     

Cucurbitane triterpenoids from Leucopaxillus gentianeus

In addition to the known bioactive triterpene cucurbitacin B (1), two new cucurbitane triterpenoids, namely, leucopaxillones A (3) and B (4), exhibiting a new oxygenation pattern among cucurbitacins, have been isolated from the mushroom Leucopaxillus gentianeus (syn. L. amarus). Cucurbitacin B (1) imparts a bitter taste to the flesh of the fungus; however, it occurs in the fruiting bodies mainly esterified as tasteless fatty acid esters 2a-c. In vitro growth inhibitory effects of compounds 1-4 on proliferation of four different human tumor cell lines (A549, CAKI-1, HepG2, MCF-7) were evaluated by using a 1-day MTT assay. Only cucurbitacin B was highly active on all lines. Free cucurbitacin B is presumed to be formed in vivo by an enzyme-mediated scission of esters 2a-c, thus constituting a chemical weapon that protects the mushrooms against parasites and predators. Compounds 1-4 are structurally different from the other few cucurbitacins isolated from Basidiomycetes, being, instead, more similar to those occurring in plants. In particular, cucurbitacin B (1) seems to represent an interesting example of secondary metabolite convergence between distant taxa such as fungi and vascular plants, where they likely exert a similar role of protection. The structures of the compounds were established by means of spectroscopic methods and X-ray diffraction on a single crystal. The absolute configuration of leucopaxillone A has been assigned on the basis of CD chirality rules.     

RASSF1A interacts with microtubule-associated proteins and modulates microtubule dynamics

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.     

Enlarged axillary nodes and position of the arms in axillary irradiation--a computed tomography and magnetic resonance imaging evaluation

The purpose of this study was to evaluate the axillary node displacement away from chest wall and their anatomical location in relation to the humeral head, according to the position of the arms, when the axilla is the site of enlarged nodes. In 13 patients with enlarged axillary nodes, the anatomical span of the nodes according to two arms positions, akimbo (A) and up over the head (U), was prospectively evaluated using computed tomography (CT) and magnetic resonance imaging (MRI). The nodes were classified into two groups, i.e. the lower and upper groups. The mean distances of the lower node group from the chest wall when the patients were in A, and U positions were 3 cm and 6.4 cm, respectively (p=0.002). The upper group nodes showed a smaller difference in the distance from the chest wall: in A position, mean 2.1 cm; in U position 2.8 cm (p=0.03). In U position, there was always a node of the lower group that was displaced in front of the humeral head. This study demonstrates the displacement of enlarged axillary nodes according to the position of the arms. In patients with axillary node involvement, CT planning should be considered when they have their arms held up over their heads.     

Immunohistochemical and molecular study of radiation-induced multiple meningiomas with pleural and pulmonary metastasis

In the present study, the telomerase activity and the putative alterations of genes involved in cell-cycle control (p53, Fas and pRb) were investigated in a radiation-induced meningioma with multiple recurrences and pleural-pulmonary metastases (the patient, a 34-year-old male, had a history of carcinoma of the tongue of testicular lymphocytic lymphoma). Expression of VEGF and vasculature pattern were also studied. Expression of VEGF, pRb and p53 were evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded samples of the tumor. VEGFmRNA was determined by competitive PCR. Fas, FasL and hTERT were evaluated by RT-PCR. Telomerase activity was examined by the TRAP assay. An intense vascularization was observed, supported by high expression of VEGFmRNA (isoforms 121 and 165). pRb and p53 were overexpressed. Fas was undetectable with PCR, whereas FasL was positive. Furthermore, the lesion showed an elevated telomerase activity (TPG, 22), according to the high expression of hTERT. These findings emphasized that even among generally benign neoplasms, such as meningiomas, some highly malignant tumors may develop, as in our case, in which several mechanisms were activated in the cancer progression to guarantee the immortalization of cellular clones (angiogenic phenomenon, activation of telomerase and of anti-apoptotic mechanisms) and the blood spread. Thus, the data illustrate the importance of searching for genetic aberrations (which are a hallmark of malignancy) in meningiomas, as predictive and reliable factors of the possibility to recur and to metastasize.     

H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization

Human thyroid papillary carcinomas are characterized by rearrangements of the RET protooncogene with a number of heterologous genes, which generate the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of a gene named H4(D10S170). We have characterized the H4(D10S170) gene product, showing that it is a ubiquitously expressed 55 KDa nuclear and cytosolic protein that is phosphorylated following serum stimulation. This phosphorylation was found to depend on mitogen-activated protein kinase (MAPK) Erk1/2 activity and to be associated to the relocation of H4(D10S170) from the nucleus to the cytosol. Overexpression of the H4(D10S170) gene was able to induce apoptosis of thyroid follicular epithelial cells; conversely a carboxy-terminal truncated H4(D10S170) mutant H4(1-101), corresponding to the portion included in the RET/PTC1 oncoprotein, behaved as dominant negative on the proapoptotic function and nuclear localization of H4(D10S170). Furthermore, conditional expression of the H4(D10S170)-dominant negative truncated mutant protected cells from stress-induced apoptosis. The substitution of serine 244 with alanine abrogated the apoptotic function of H4(D10S170). These data suggest that loss of the H4(D10S170) gene function might have a role in thyroid carcinogenesis by impairing apoptosis.