Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.

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Cure for ductal adenocarcinoma of the pancreas is restricted to resectable tumors, but survival after surgery is still poor. Despite apparently curative resection, these cancers rapidly recur. Thus, the present pathologic examination should be enriched by sensitive methods to detect minimal residual disease. In a prospective setting we studied the frequency of minimal residual disease after curative resection by routine histopathology, immunohistology, and polymerase chain reaction (PCR) for mutated K-ras. Furthermore, the prognostic implication of detecting of MRD was determined. Prospectively, tumor tissue and corresponding paraaortic lymph nodes were obtained from 78 patients, who underwent surgery for pancreatic head tumors between 1999 and 2001. Sixty-nine of 78 cases were diagnosed for ductal adenocarcinoma (study group), whereas nine cases were diagnosed for benign pancreatic tumors (control group). Paraaortic lymph nodes were examined in step sections by routine histopathology (hematoxylin and eosin) and immunohistology using a pan-cytokeratin antibody. DNA of the primary tumor and corresponding paraaortic lymph nodes were analyzed by PCR-based assays with respect to mutated K-ras in codon 12. The recurrence-free survival and overall survival were correlated with the results of the latter methods. In 3 of 69 patients tumor cells were detected in paraaortic lymph nodes by routine histopathology and in 5 of 69 patients by immunohistology. K-ras mutations were detected in 42 of 69 ductal adenocarcinomas (61%), whereas 12 (17%) were positive in paraaortic lymph nodes. All of the latter patients had recurrence after surgery and a significant poorer survival than those without mutated K-ras. Furthermore, paraaortic lymph nodes diagnosed for K-ras mutation were independent prognostic markers in multivariate analysis. In the control group K-ras mutations were detected in one adenoma of Vater's papilla but not in paraaortic lymph nodes. Tumor cell DNA can be detected more sensitively by the described PCR method than with hematoxylin and eosin or immunohistologic staining, leading to a higher sensitivity for detection of micrometastases. The described PCR method clearly determines subgroups of patients after curative resection with early recurrence and poor survival and could therefore enrich the pathologic examination.