Future studies may support our hypothesis that the pretreatment serum level of MMP-9 is a new powerful prognostic marker and can help to stratify NSCLC patients with stage I/II disease into low- and high-risk groups.

A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.

The clinical relevance of the carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, squamous cell carcinoma antigen (SCC), thymidine kinase (TK), and deoxythymidine-5'-triphosphatase (dTTPase) as tumor markers in the diagnosis and follow-up treatment of 26 patients with head and neck cancer is evaluated. Serum levels prior to treatment were found elevated just above the upper limit of normal in 46% (SCC), 15% (CEA), 12% (CA 19-9), 27% (TK), and 39% (dTTPase) of all patients. If all markers were taken into account, they were elevated in 73% of the untreated patients. However, only in a few cases were the tumor marker values elevated significantly (8%-12%). No significant correlation was detected between serum levels and tumor localization, staging, grading, or performance status for any of the markers. In the follow-up none of the markers tested revealed any disease-related information despite therapy variation. Patients with originally elevated marker levels showed decreasing and in some cases increasing values after primary therapy, although no tumor recurrence was detected. Even considering the results as preliminary due to the rather small sample size, they suggest that the routine assessment of CEA, CA 19-9, SCC, TK, and dTTPase serum levels is of limited practical value.

Thymidine triphosphate monophosphohydrolase (dTTPase), an enzyme which catalyzes the hydrolysis of dTTP to the corresponding diphosphate (dTDP), has been purified to homogeneity from human serum. The enzyme sediments with 3.8 S in sucrose density gradients. A Stokes radius of 31 A is estimated by gel filtration. Accordingly, its molecular weight is 48 500. Since only one single band of Mr 24 000 is detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the native enzyme seems to be composed of two identical subunits. The enzyme exhibits optimal activity over a pH range from 7 to 9, and the activation energy is estimated to be 7.1 kcal/mol (29.7 kJ/mol) at pH 7.8. While the enzyme is active in the absence of added divalent cations, the activity can be inhibited by ethylenediaminetetracetic acid (EDTA) but not by phenanthroline. The inhibition caused by EDTA is reversed by Mn2+. Zn2+ causes a complete inhibition of enzyme activity. No requirement exists for a sulfhydryl compound. The enzyme has an Rf value of 0.45, an isoelectric point of 5.2, and an apparent Km value of 40 microM for dTTP. dUTP and UTP are degraded by about 50 and 20% of the rate of dTTP hydrolysis, respectively. Other deoxyribonucleosides or ribonucleoside triphosphates do not serve as substrates for the dTTPase. The existence of this enzyme is significant since it could play a role in the regulation of the cellular dTTP levels.

A photometric method for the determination of the acid nucleoside triphosphatase (EC 3.6.1.-) is described, in which inorganic phosphate is liberated from ATP or other nucleoside triphosphates. Colorimetric determination of liberated phosphate is based on the formation of a green complex of phosphomolybdate and malachite green hydrochloride. Optimal test conditions were evaluated as well as the sample preparation. The enzyme activities measured in 100 normal human sera are in the range of 0.5 to 9.0 U/l with an average of 4.0 U/l for men and 3.8 U/l for women.