Melissa G. Steiner

HIV-1 upregulates Fas ligand expression in CD4+ T cells in vitro and in vivo: association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid.

CD4+ T‐lymphocyte apoptosis has been associated with human immunodeficiency virus (HIV)‐1 infection in vitro, paralleling the expression of Fas (APO‐1, CD95) on peripheral blood mononuclear cells from patients with HIV disease. However, the link between Fas induction, T‐cell activation, and cell death is unclear. We document, for the first time, marked upregulation of expression of mRNA for the ligand for Fas in peripheral blood mononuclear cells from HIV seropositive individuals, and demonstrate the ability of HIV infection to induce such expression in CD4+ T cells in vitro. We also define the relevance of this expression to HIV‐mediated CD4+ T cell death. Our ability to downregulate Fas ligand message and suppress HIV‐mediated apoptosis with aurintricarboxylic acid, a clinically used protease inhibitor with known activity against programmed cell death in other systems, may open up a new area of therapy for HIV infection.

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 – 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 – 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.

Characterization of Novel Cell Lines From Pleomorphic Adenomas of the Parotid Gland Established in a Collagen Gel System

The pathobiology of salivary neoplasms can best be studied in a model system that reflects the native state of the tumor. The present study describes the use of a three‐dimensional collagen gel (organoid) system in which pleomorphic adenomas of the parotid gland were propagated in vitro. Five pleomorphic adenoma cultures were established as organoid gels and compared with touch‐preparations or cryopreserved specimens of native tumor. The organoid cultures demonstrated normal DNA content, the expression of myoepithelial cell proteins, and the production of sulfated acid mucins; these cellular and secretory features mimicked those found in the archival specimens. Further, organoid cultures of pleomorphic adenoma could be initiated after monolayer culture, demonstrating that culture on a plastic support does not alter the nature of the cells. Development of an in vitro culture system that maintains the native state of pleomorphic adenoma is an important tool for studying the pathobiology of these tumors.

Poster 32 Characterization of Novel Cell Lines from Pleomorphic Adenomas of the Parotid Gland Established in a Collagen Gel System

The pathobiology of salivary neoplasms can best be studied in a model system that reflects the native state of the tumor. The present study describes the use of a three-dimensional collagen gel (organoid) system in which pleomorphic adenomas of the parotid gland were propagated in vitro. Five pleomorphic adenoma cultures were established as organoid gels and compared with touch-preparations or cryopreserved specimens of native tumor. The organoid cultures demonstrated normal DNA content, the expression of myoepithelial cell proteins, and the production of sulfated acid mucins; these cellular and secretory features mimicked those found in the archival specimens. Further, organoid cultures of pleomorphic adenoma could be initiated after monolayer culture, demonstrating that culture on a plastic support does not alter the nature of the cells. Development of an in vitro culture system that maintains the native state of pleomorphic adenoma is an important tool for studying the pathobiology of these tumors.

Poster 30 Expression of Very Late Antigen (β1) Integrins in Pleomorphic Adenoma Cell Lines

ations in pleomorphic adenomas at microsatellite loci in regions reported to be sites of cytogenetic abnormalities. A polymerase chain reaction-based single-strand conformation polymorphism analysis was used to identify loss of heterozygosity (LOH) and microsatellite instability in pleomorphic adenoma specimens. DNA extracted from 36 freshfrozen paired normal and tumor specimens from 18 patients (18 normal, 14 pleomorphic adenoma, and four pleomorphic adenoma with a foci of carcinoma ex pleomorphic adenoma) was analyzed for microsatellite alterations at loci on chromosomes 3p, 6q, 8p, and 8q. Correlation with clinical and pathologic features was performed. Overall, 7 of 18 cases (39%) manifested LOH at the loci tested. LOH was noted on 3p, 6q, 8p, and 8q microsatellite loci in 5.6%, 13.0%, 15%, and 27% of informative cases, respectively. No microsatellite instability was noted at the loci analyzed. Specimens from patients with carcinoma e x pleomorphic adenoma did not manifest enhanced LOH. We observed a lack of association between chromosomal abnormalities and age, sex, tumor size, histologic features, and DNA ploidy in these tumors. A subset of pleomorphic adenomas exhibited loss of heterozygosity at microsatellite loci on 3p, 6q, 8p, and 8q. The incidence did not appear to be increased in patients with focal carcinoma ex pleomorphic adenoma, indicating that LOH at these loci is an early event in tumorigenesis. In addition, our results indicate that genetic alterations other than translocations are involved in the genesis of these neoplasms. Further evaluation at these loci is needed to identify potential tumor suppressor genes that function in initiation and progression of pleomorphic adenomas.