Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill transformed cells. We have studied the expression and functionality of the TRAIL apoptotic pathway in Ewing's sarcoma. We demonstrate that tumors from patients with Ewing's sarcoma express receptors TRAIL-R1 and -R2. Using a panel of nine Ewing's sarcoma cell lines TRAIL could induce apoptosis in seven cell lines. Preincubation with interferon-gamma rendered the two resistant cell lines sensitive. TRAIL was the most potent inducer of apoptosis when compared to Fas ligand or TNF. TRAIL-mediated apoptosis could be inhibited by various caspase-inhibitors. No difference in the surface expression of TRAIL-receptors was observed between sensitive and resistant cell lines. Also, all cell lines had similar levels of expression of Flice-like inhibitory protein (FLIP) on immunoblot. However, the two resistant cell lines had only very low level expression of caspase 8 on RNA and protein level. In summary, we show that Ewing's sarcoma expresses receptors for TRAIL, and that cells are exquisitely sensitive to TRAIL-mediated apoptosis. These results may warrant clinical trials with TRAIL in Ewing's sarcoma once the safety of TRAIL for humans has been established.

Gene expression of the hematopoietic cell phosphatase in juvenile myelomonocytic leukemia.

We analyzed the relative expression of Hematopoietic cell phosphatase (HCP) in mononuclear cells (MNC) of peripheral blood (PB), bone marrow (BM) and spleen of patients with juvenile myelomonocytic leukemia (JMML) and normal donors. Two regions of HCP with alternative exon skipping of exon 6 or exon 12 are described. There was no difference in the expression of the amplified HCP cDNA regions in MNC of JMML patients compared to normal donors. The two forms of exon skipping were present in unstimulated MNC of JMML patients or normal donors. In contrast, phytohemagglutinin (PHA) stimulated MNC of normal donors, Epstein-Barr Virus (EBV) transformed B-cells of JMML patients, BFU-E and CFU-GM derived colonies of JMML patients, and the cell lines K562 and HEL did not or only barely express these two forms of exon skipping. These results may indicate that alternative HCP exon skipping may be associated with the proliferative state of the cell.

GADD45 induction of a G2/M cell cycle checkpoint.

G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.

Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin.

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.

Simultaneous expression of Fas and nonfunctional Fas ligand in Ewing's sarcoma.

Fas-Fas ligand interactions play a central role in the regulation of the immune response. Fas ligand expression by tumors has been implicated in the abrogation of the host antitumor response by killing of Fas-positive effector lymphocytes. We have studied the presence and functional status of Fas and Fas ligand in Ewing's sarcoma. All Ewing's sarcoma cell lines tested expressed Fas on their surface. Three of the cell lines were readily killed after ligation of the Fas receptor. Four additional cell lines exhibited Fas-mediated apoptosis after preincubation with IFN-gamma and/or cycloheximide, whereas two cell lines were resistant to Fas-mediated killing. With regard to Fas ligand, all cell lines examined were positive for protein by immunoblot, and specificity was confirmed by reverse transcription-PCR. However, using flow cytometric analysis, Fas ligand could only be detected in Ewing's sarcoma cells after permeabilization. Furthermore, the cell lines were not capable of inducing apoptosis of Fas-sensitive Jurkat cells. In addition, Ewing's sarcoma cell lines were able to serve as stimulators for the generation of cytotoxic effector lymphocytes and were susceptible to lysis by them. Therefore, Fas ligand is expressed in Ewing's sarcoma but is not functional, suggesting that Ewing's sarcoma is a potential target for immunotherapy.

Multilocular thymic cysts in children with human immunodeficiency virus infection: clinical and pathologic aspects.

BACKGROUND: Children with human immunodeficiency virus (HIV) infection have an increased susceptibility to severe and unusual infections, malignancies, and disorders characterized by abnormal lymphoproliferation (e.g., lymphoid interstitial pneumonitis). We report a novel disease entity associated with pediatric HIV infection that is characterized by massive enlargement of the thymus as a result of lymphoid hyperplasia and multicystic changes. METHODS: Eight patients with HIV infection and cystic enlargement of the thymus are subject of this report. The status of their HIV disease and its clinical and radiologic manifestations at the time of diagnosis of the mediastinal mass are described. Tissue specimens were obtained from six patients and examined by microscopy and immunohistochemistry. The specimens were also evaluated for the evidence of HIV and Epstein-Barr virus by in situ hybridization. RESULTS: Patients were between 2.1 and 12.1 years of age, with CD4+ cell counts between 102 and 733 cells/mm3. In all eight cases an anterior mediastinal mass was discovered incidentally on radiography of the chest, and computed tomography of the chest revealed a multicystic appearance. Histologic examination demonstrated distortion of the thymic architecture by focal cystic changes, lymphoid follicular hyperplasia, diffuse plasmacytosis, and multinucleated giant cells. In situ hybridization revealed HIV particles on the surface of follicular dendritic cells. Further, results of in situ hybridization for EBV were positive in lymphoid cells from biopsy samples of four patients. The patients were followed between 8 months and 4.8 years. In five patients the mass either decreased in size or resolved completely. CONCLUSIONS: We describe a series of children with HIV infection and multilocular thymic cysts. We hypothesize that aberrant immunoregulation in these HIV-infected children leads to follicular hyperplasia and multicystic changes in the thymus, causing massive enlargement. EBV infection might also contribute to the pathogenesis of this process. Because none of our patients had symptoms from the mass, and there was no evidence of malignancy in the examined biopsy samples, it seems prudent to manage such children with careful follow-up examinations.

The p53-regulated cyclin G gene promotes cell growth: p53 downstream effectors cyclin G and Gadd45 exert different effects on cisplatin chemosensitivity.

Among the p53-regulated genes that have been identified thus far, cyclin G is a relatively recent one. We conducted a series of experiments aimed at elucidating cyclin G function. Ectopic overexpression of cyclin G in human RKO colon carcinoma cells accelerated cell growth. Transfection of normal human fibroblasts with the cyclin G expression vector promoted clonal expansion. Cyclin G immune complexes isolated from the transfected cells exhibited appreciable levels of cyclin-dependent kinase activity, as evidenced using histone H1 as a substrate. The retinoblastoma protein, pRb, was detectable in cyclin G immune complexes, raising the possibility that Rb may be one mediator of cyclin G action. Cyclin G-overexpressing cells were more sensitive to cisplatin cytotoxicity than the parent cells, probably because cyclin G overexpression overrides cell cycle checkpoint(s). Overexpression of another p53-regulated gene, GADD45, by contrast, protected cells from cisplatin killing. These findings suggest that different downstream effectors of the p53 pathway may exert different effects on cellular survival after treatment with cancer chemotherapy drugs such as cisplatin.

Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin.

Loss of p53 function in cancer cells commonly results in a condition of genomic instability. This is believed to emanate from a loss of the G1 checkpoint response to DNA damage. While the role of p53 in the induction of a G1 arrest is well-accepted, additional p53 functions are being discovered. Cell cycle checkpoints presumably function to allow additional time for DNA repair after damage is incurred, however, genetic studies in yeast suggest that components of the checkpoint pathway may also be involved in DNA lesion processing (Lydall and Weinert, 1995). Recent evidence suggests that this may also be the case for p53, as suggested by numerous reports linking p53 function to DNA repair. Thus, loss of p53 function might contribute to genomic instability independent of G1-arrest. In the present study, we explored the effect of p53 disruption and consequences of antisense GADD45 expression on the DNA repair capacity of human colon carcinoma RKO cells. DNA repair was assayed using host-cell reactivation of u.v.-damaged reporter plasmids and unscheduled DNA synthesis experiments in transiently-transfected cells. We show that a number of transfected genes that suppress p53 function reduce the ability of cells to repair u.v.-induced DNA damage. Moreover, cells in which expression of the p53-regulated gene GADD45 was blocked by antisense vectors, also showed altered levels of DNA repair. Blocking Gadd45 expression by constitutive antisense expression sensitized cells to killing by u.v.-radiation or by cis-platinum (II) diamine-dichloride (CDDP, or cisplatin), a cancer chemotherapy drug which produces DNA cross-links. These findings suggest the involvement of downstream effectors of the p53 pathway in the coordination of cell cycle arrest and DNA repair.

Multilocular thymic cysts: imaging features in children with human immunodeficiency virus infection.

PURPOSE: To evaluate the radiologic and follow-up features of multilocular thymic cysts in children with human immunodeficiency virus (HIV) infection. MATERIALS AND METHODS: Four HIV-infected children with large anterior mediastinal masses depicted at routine chest radiography underwent ultrasonography (US), unenhanced and contrast material-enhanced computed tomography (CT), and unenhanced and gadolinium-enhanced MR imaging of the chest. Gallium scanning was also performed in three of the four children. The patients underwent follow-up radiologic examinations for 8-15 months. RESULTS: The multiloculated nature of the masses was depicted at contrast-enhanced but not unenhanced CT. Similarly, the septations were depicted on T2-weighted, short inversion time inversion-recovery (STIR), and contrast-enhanced T1-weighted, MR images but not on the unenhanced T1-weighted images. US scans depicted the septations within each mass, but findings were technically limited because only portions of each mass were depicted. Gallium scans in three masses depicted uptake of radionuclide in two and no uptake in one. Surgical biopsy was performed in each mass: Follicular hyperplasia and diffuse plasmacytosis of the thymus were found but not evidence of neoplastic or infectious origin. At follow-up, the mass decreased in volume in two patients, did not change in one patient, and increased in volume in one patient. CONCLUSION: HIV-infected patients with asymptomatic mediastinal masses depicted at routine chest radiography should undergo contrast-enhanced CT. If a solid mass is depicted, biopsy should be performed to exclude neoplastic or infectious origins. If a multiloculated anterior mediastinal mass is depicted, symptomatic follow-up is adequate since the finding represents a rare multilocular thymic cyst that does not have negative clinical implications.