Effect of Standard Radiotherapy With Cisplatin vs Accelerated Radiotherapy With Panitumumab in Locoregionally Advanced Squamous Cell Head and Neck Carcinoma: A Randomized Clinical Trial.

IMPORTANCE: The Canadian Cancer Trials Group study HN.6 is the largest randomized clinical trial to date comparing the concurrent administration of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies with radiotherapy (RT) to standard chemoradiotherapy in locoregionally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). OBJECTIVE: To compare progression-free survival (PFS) in patients with LA-SCCHN treated with standard-fractionation RT plus high-dose cisplatin vs accelerated-fractionation RT plus the anti-EGFR antibody panitumumab. DESIGN, SETTING, AND PARTICIPANTS: A randomized phase 3 clinical trial in 17 Canadian centers. A total of 320 patients were randomized between December 2008 and November 2011. INTERVENTIONS: Patients with TanyN+M0 or T3-4N0M0 LA-SCCHN were randomized 1:1 to receive standard-fractionation RT (70 Gy/35 over 7 weeks) plus cisplatin at 100 mg/m2 intravenous for 3 doses (arm A) vs accelerated-fractionation RT (70 Gy/35 over 6 weeks) plus panitumumab at 9 mg/kg intravenous for 3 doses (arm B). MAIN OUTCOMES AND MEASURES: Primary end point was PFS. Due to an observed declining event rate, the protocol was amended to a time-based analysis. Secondary end points included overall survival, local and regional PFS, distant metastasis-free survival, quality of life, adverse events, and safety. RESULTS: Of 320 patients randomized (268 [84%] male; median age, 56 years), 156 received arm A and 159 arm B. A total of 93 PFS events occurred. By intention-to-treat, 2-year PFS was 73% (95% CI, 65%-79%) in arm A and 76% (95% CI, 68%-82%) in arm B (hazard ratio [HR], 0.95; 95% CI, 0.60-1.50; P = .83). The upper bound of the HR 95% CI exceeded the prespecified noninferiority margin. Two-year overall survival was 85% (95% CI, 78%-90%) in arm A and 88% (95% CI, 82%-92%) in arm B (HR, 0.89; 95% CI, 0.54-1.48; P = .66). Incidence of any grade 3 to 5 nonhematologic adverse event was 88% in arm A and 92% in arm B (P = .25). CONCLUSIONS AND RELEVANCE: With a median follow-up of 46 months, the PFS of panitumumab plus accelerated-fractionation RT was not superior to cisplatin plus standard-fractionation RT in LA-SCCHN and noninferiority was not proven. Despite having negative results, HN.6 has contributed important data regarding disease control and toxic effects of these treatment strategies. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00820248.

Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.

A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity.

MAP kinases from bovine brain: purification and characterization.

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.

Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells.

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.

Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.

Regression of cardiac hypertrophy in spontaneously hypertensive rats by enalapril and the expression of contractile proteins.

Several experimental models involving the development of cardiac hypertrophy in adult rats are characterized by the reexpression of the fetal isoform of myosin heavy chain (V3). To determine whether a similar adult-to-fetal shift in the expression of the thin-filament proteins occurs during cardiac hypertrophy, we have examined the expression of the isoforms of myosin, tropomyosin, and troponin T in the left ventricle of young spontaneously hypertensive rats (SHR) with and without treatment using enalapril, an angiotensin converting enzyme inhibitor. Phosphorylation of tropomyosin, which is predominant in the fetal state, was also analyzed. Twelve-week-old SHR were treated with enalapril for 2, 5, 8, and 9 weeks followed by withdrawal of treatment for 9 weeks. Control SHR, without drug treatment, were weight- and age-matched. After 9 weeks of enalapril treatment, mean arterial blood pressure was reduced (from 166 +/- 11 to 89 +/- 5 mm Hg), and left ventricular weight/body weight ratio was regressed (from 2.53 +/- 0.14 to 1.96 +/- 0.05 g/kg) to normotensive levels. During the 9-week treatment period, the percent V3 decreased in SHR substantially from 35 +/- 3% to 13 +/- 1%. There was a significant correlation between the left ventricular hypertrophy and the percent V3 myosin expression in the SHR during regression (r = 0.697, p less than 0.001). However, only the adult isoforms of tropomyosin and troponin T were detected in the SHR with or without enalapril treatment, and the level of tropomyosin phosphorylation remained constant irrespective of the degree of left ventricular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)