Faculty Bibliography

PURPOSE: Extracellular matrix remodeling during tumor growth plays an important role in angiogenesis. Our preclinical data suggest that a newly identified cryptic epitope (HU177) within collagen type IV regulates endothelial and melanoma cell adhesion in vitro and angiogenesis in vivo. In this study, we investigated the clinical relevance of HUI77 shedding in melanoma patient sera. EXPERIMENTAL DESIGN: Serum samples from 291 melanoma patients prospectively enrolled at the New York University Medical Center and 106 control subjects were analyzed for HU177 epitope concentration by a newly developed sandwich ELISA assay. HU177 serum levels were then correlated with clinical and pathologic parameters. RESULTS: Mean HU177 epitope concentration was 5.8 ng/mL (range, 0-139.8 ng/mL). A significant correlation was observed between HU177 concentration and nodular melanoma histologic subtype [nodular, 10.3 +/- 1.6 ng/mL (mean +/- SE); superficial spreading melanoma, 4.5 +/- 1.1 ng/mL; all others, 6.1 +/- 2.1 ng/mL; P = 0.01 by ANOVA test]. Increased HU177 shedding also correlated with tumor thickness (< or =1.00 mm, 3.8 +/- 1.1 ng/mL; 1.01-3.99 mm, 8.7 +/- 1.3 ng/mL; > or =4.00 mm, 10.3 +/- 2.4 ng/mL; P = 0.003 by ANOVA). After multivariate analysis controlling for thickness, the correlation between higher HU177 concentration and nodular subtype remained significant (P = 0.03). The mean HU177 epitope concentration in control subjects was 2.4 ng/mL. CONCLUSIONS: We report that primary melanoma can induce detectable changes in systemic levels of cryptic epitope shedding. Our data also support that nodular melanoma might be biologically distinct compared with superficial spreading type melanoma. As targeted interventions against cryptic collagen epitopes are currently undergoing phase I clinical trial testing, these findings indicate that patients with nodular melanoma may be more susceptible to such targeted therapies

Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.

Transient receptor potential (TRP) ion channels have been implicated in detecting chemical, thermal, and mechanical stimuli in organisms ranging from mammals to Caenorhabditis elegans. It is well established that TRPA1 detects and mediates behavioral responses to chemical irritants. However, the role of TRPA1 in detecting thermal and mechanical stimuli is controversial. To further clarify the functions of TRPA1 channels in vertebrates, we analyzed their roles in zebrafish. The two zebrafish TRPA1 paralogs are expressed in sensory neurons and are activated by several chemical irritants in vitro. High-throughput behavioral analyses of trpa1a and trpa1b mutant larvae indicate that TRPA1b is necessary for behavioral responses to these chemical irritants. However, TRPA1 paralogs are not required for behavioral responses to temperature changes or for mechanosensory hair cell function in the inner ear or lateral line. These results support a role for zebrafish TRPA1 in chemical but not thermal or mechanical sensing, and establish a high-throughput system to identify genes and small molecules that modulate chemosensation, thermosensation, and mechanosensation.

NAD(P)H:quinone oxidoreductase 1 null (NQO1(-/-)) mice exposed to 3 Gy of gamma-radiation showed an increase in neutrophils, bone marrow hypercellularity, and enlarged lymph nodes and spleen. The spleen showed disrupted follicular structure, loss of red pulp, and granulocyte and megakarocyte invasion. Blood and histologic analysis did not show any sign of infection in mice. These results suggested that exposure of NQO1(-/-) mice to gamma-radiation led to myeloproliferative disease. Radiation-induced myeloproliferative disease was observed in 74% of NQO1(-/-) mice as compared with none in wild-type (WT) mice. NQO1(-/-) mice exposed to gamma-radiation also showed lymphoma tissues (32%) and lung adenocarcinoma (84%). In contrast, only 11% WT mice showed lymphoma and none showed lung adenocarcinoma. Exposure of NQO1(-/-) mice to gamma-radiation resulted in reduced apoptosis in granulocytes and lack of induction of p53, p21, and Bax. NQO1(-/-) mice also showed increased expression of myeloid differentiation factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and Pu.1. Intriguingly, exposure of NQO1(-/-) mice to gamma-radiation failed to induce C/EBPalpha and Pu.1, as was observed in WT mice. These results suggest that decreased p53/apoptosis and increased Pu.1 and C/EBPalpha led to myeloid hyperplasia in NQO1(-/-) mice. The lack of induction of apoptosis and differentiation contributed to radiation-induced myeloproliferative disease in NQO1(-/-) mice.

PURPOSE: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of (12)C and (20)Ne ions. MATERIALS AND METHODS: Chinese hamster ovary (CHO-K1) cells were exposed to high LET (12)C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and (20)Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up. RESULTS: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440-830 keV/microm for (12)C and for 1020-1600 keV/microm for (20)Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET. CONCLUSIONS: The RBE decreases with increasing LET in the range between 440 and 1600 keV/microm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.

To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of (65)Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1,000 k, 45 k and 11 k) plus one of 110 kDa. (65)Cu (141 microg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. (63)Cu/(65)Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.

Egg-laying behavior of the Caenorhabditis elegans hermaphrodite is regulated by G protein signaling pathways. Here we show that the egg laying-defective mutant egl-6(n592) carries an activating mutation in a G protein-coupled receptor that inhibits C. elegans egg-laying motor neurons in a G(o)-dependent manner. Ligands for EGL-6 are Phe-Met-Arg-Phe-NH(2) (FMRFamide)-related peptides encoded by the genes flp-10 and flp-17. flp-10 is expressed in both neurons and non-neuronal cells. The major source of flp-17 peptides is a pair of presumptive sensory neurons, the BAG neurons. Genetic analysis of the egl-6 pathway revealed that the EGL-6 neuropeptide signaling pathway functions redundantly with acetylcholine to inhibit egg-laying. The retention of embryos in the uterus of the C. elegans hermaphrodite is therefore under the control of a presumptive sensory system and is inhibited by the convergence of signals from neuropeptides and the small-molecule neurotransmitter acetylcholine

The outcomes of 20 patients diagnosed with osteoarthritis or rheumatoid arthritis with body mass index less than 18.5 (considered underweight) who received total hip arthroplasty at a single institution were reviewed. Surgical complications in the first 30 days after surgery included 1 prolonged surgical site drainage and 3 posterior dislocations. Two patients experienced medical complications that included hematemesis, confusion, aspiration pneumonia, and death. Sixty-five percent of the patients received at least one blood transfusion. Harris hip scores improved from 35 to 81 (P < .05) at an average of 6.1 years (2-10.1 years) of follow-up. Total hip arthroplasty is effective in patients who are underweight; however, they appear to be at an increased risk of dislocation and blood transfusion

The strengths of 3 hip spacer constructs--Steinmann pins, a short intramedullary nail (both cement-incorporated), and a Charnley prosthesis--were determined and compared with the strength of a commercially available hip spacer. The hip prosthesis construct was more than twice as strong as the other 2 constructs and was equivalent in strength to the commercial spacer. For spacer applications in which limited weight-bearing is anticipated, the hip prosthesis construct appears more efficacious, but its pros and cons should be compared with those of the commercial product

OBJECTIVES: An epithelial ovarian cancer cell line constitutively expressing the androgen receptor was created to evaluate the mechanism and effects of androgen receptor activation on epithelial ovarian cancer cell invasion. METHODS: Immunocytochemistry and Western blot analyses confirmed androgen receptor expression. Boyden chamber invasion assays were performed using cells treated with the androgen receptor ligands medroxyprogesterone acetate or dihydrotestosterone. The matrix metalloproteinases associated with invasion were investigated using zymographic assays. RESULTS: Androgen receptor-mediated invasion is ligand dependent. While both medroxyprogesterone acetate and dihydrotestosterone signal through androgen receptor, medroxyprogesterone acetate is more effective at stimulating invasion of epithelial ovarian cancer cells. Unlike the wild-type epithelial ovarian cancer cells, this increase in invasion in androgen receptor + epithelial ovarian cancer cells does not seem to be dependent on matrix metalloproteinase 2 or 9 activation. CONCLUSION: Although classified as a progestin, medroxyprogesterone acetate has significant androgenic activity unique from the pure androgen dihydrotestosterone. Our studies suggest that pharmacologic doses of medroxyprogesterone acetate may actually increase the invasive potential of epithelial ovarian cancer cells.