The identification of 15 N-labeled 3-nitrotyrosine (NTyr) by gas chromatography͞mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15 N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of proteinbound NTyr in normal rat plasma to be Ϸ0-1 residues per 10 6 Tyr with a detection limit of 0.5 per 10 7 Tyr when >100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an Ϸ6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N G -monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to Ϸ13 residues per 10 6 Tyr, an Ϸ40-fold elevation compared with plasma protein of untreated rats; cotreatment with N Gmonomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO x exposure such as inflammation and smoking.

Objectives: : To determine expression of cell cycle and apoptotic genes, biochemical analysis of CCL23 and antisense cyclin D1-transfected CCL23 (CCL23AS) cells in the presence of cisplatin was performed. In addition, biochemical analysis of CAL27 cells before and after treatment with cisplatin was performed to determine expression of cell cycle genes. Design: CCL23, CCL23AS, and CAL27 cell lines were treated with cisplatin. Western blot analysis, fluorescenceactivated cell sorting, and apoptosis assays were performed. Setting: In vitro study of head and neck cancer cell lines CCL23, CCL23AS, and CAL27. Intervention: CCL23, CCL23AS, and CAL27 cells were treated with cisplatin. Main Outcome Measures: Expression of p16, p21, p53, Bcl-xL, Bcl-xS, p27, DP1, MDM2, Bcl-2, c-Jun, and Jun-D were assessed using Western blot analysis. Results: There was increased expression of p16, p21, p53, BCLxL, and BCLxS genes with cisplatin treatment in the

B-3b.

Pretreatment of HNSCC cells with long-acting NO donors enhances cisplatin activity. Short- and medium-acting NO donors do not exert a toxic effect and do not augment the activity of cisplatin. NO agonists should be considered in the future as a possible adjunct to cisplatin in the treatment of HNSCC. Further studies with animal models are necessary to further clarify this relationship.

Background: We have previously described our treatment algorithm for patients with small head and neck cancers with advanced cervical metastases (stage N2 or greater). Primary radiotherapy is given to the primary site and neck, followed 6 weeks later with endoscopy and biopsy of the primary site. If biopsy of the primary site is negative by frozen section, an immediate neck dissection is performed even when no clinical residual neck disease is present. Our initial review found that 36% of patients with a complete clinical response to radiotherapy had positive nodes on histological examination. Study Design: Retrospective. Methods: The medical records of 71 patients treated at UCLA Medical Center from 1986 to 1999 by this algorithm were reviewed. Results: After radiotherapy, 69 of 71 patients had a complete response at their primary site. Forty-two patients had a complete clinical response in the neck. Seventy-one neck dissections were performed. Overall, 31 of 71 neck dissections (44%) had positive nodes. Among the 42 patients with a complete response to radiotherapy, 13 (31%) had positive histological nodes. Among the 29 patients with a partial response to radiotherapy, 17 (59%) had positive nodes. Follow-up and incidence of neck recurrence are discussed. Conclusion: Planned neck dissection for advanced cervical metastases remains controversial for patients with a complete clinical response to radiotherapy. However, our results suggest that clinical assessment after radiotherapy cannot assure the absence of neck disease. Until there are reliable methods to distinguish which patients are truly free of neck disease, we believe the benefits of a planned neck dissection outweigh the low morbidity of this procedure.