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<p>Glutathione transferases (GSTs) are detoxication enzymes involved in the cellular protection against a wide range of reactive substances. The role of GSTs is to catalyze the conjugation of glutathione with electrophilic compounds, which generally results in less toxic products. </p><p>The ability to catalyze the denitrosation of the anticancer drug 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU) was measured in twelve different GSTs. Only three of the enzymes showed any measurable activity with BCNU, of which human GST T1-1 was the most efficient. This is of special interest, since human GST T1-1 is a polymorphic protein and its expression in different patients may be crucial for the response to BCNU.</p><p>DNA shuffling was used to create a mutant library by recombination of cDNA coding for two different Theta-class GSTs. In total, 94 randomly picked mutants were characterized with respect to their catalytic activity with six different substrates, expression level and sequence. A clone with only one point mutation compared to wild-type rat GST T2-2 had a significantly different substrate-activity pattern. A high expressing mutant of human GST T1-1 was also identified, which is important, since the yield of the wild-type GST T1-1 is generally low. </p><p>Characterization of the Theta library demonstrated divergence of GST variants both in structure and function. The properties of every mutant were treated as a point in a six-dimensional substrate-activity space. Groups of mutants were formed based on euclidian distances and K-means cluster analyses. Both methods resulted in a set of five mutants with high alkyltransferase activities toward dichloromethane and 4-nitrophenethyl bromide (NPB). </p><p>The five selected mutants were used as parental genes in a new DNA shuffling. Addition of cDNA coding for mouse and rat GST T1-1 improved the genetic diversity of the library. The evolution of GST variants was directed towards increased alkyltransferase activity including activity with the anticancer drug BCNU. NPB was used as a surrogate substrate in order to facilitate the screening process. A mutant from the second generation displayed a 65-fold increased catalytic activity with NPB as substrate compared to wild-type human GST T1-1. The BCNU activity with the same mutant had increased 175-fold, suggesting that NPB is a suitable model substrate for the anticancer drug. Further evolution presented a mutant in the fifth generation of the library with 110 times higher NPB activity than wild-type human GST T1-1.</p>

Glutathione transferases (GSTs) are detoxication enzymes involved in the cellular protection against a wide range of reactive substances. The role of GSTs is to catalyze the conjugation of glutathione with electrophilic compounds, which generally results in less toxic products. The ability to catalyze the denitrosation of the anticancer drug 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU) was measured in twelve different GSTs. Only three of the enzymes showed any measurable activity with BCNU, of which human GST T1-1 was the most efficient. This is of special interest, since human GST T1-1 is a polymorphic protein and its expression in different patients may be crucial for the response to BCNU. DNA shuffling was used to create a mutant library by recombination of cDNA coding for two different Theta-class GSTs. In total, 94 randomly picked mutants were characterized with respect to their catalytic activity with six different substrates, expression level and sequence. A clone with only one point mutation compared to wild-type rat GST T2-2 had a significantly different substrate-activity pattern. A high expressing mutant of human GST T1-1 was also identified, which is important, since the yield of the wild-type GST T1-1 is generally low. Characterization of the Theta library demonstrated divergence of GST variants both in structure and function. The properties of every mutant were treated as a point in a six-dimensional substrate-activity space. Groups of mutants were formed based on euclidian distances and K-means cluster analyses. Both methods resulted in a set of five mutants with high alkyltransferase activities toward dichloromethane and 4-nitrophenethyl bromide (NPB). The five selected mutants were used as parental genes in a new DNA shuffling. Addition of cDNA coding for mouse and rat GST T1-1 improved the genetic diversity of the library. The evolution of GST variants was directed towards increased alkyltransferase activity including activity with the anticancer drug BCNU. NPB was used as a surrogate substrate in order to facilitate the screening process. A mutant from the second generation displayed a 65-fold increased catalytic activity with NPB as substrate compared to wild-type human GST T1-1. The BCNU activity with the same mutant had increased 175-fold, suggesting that NPB is a suitable model substrate for the anticancer drug. Further evolution presented a mutant in the fifth generation of the library with 110 times higher NPB activity than wild-type human GST T1-1.

Objective: To assess the safety and effectiveness of bis-chloroethylnitrosourea (BCNU) wafers in elderly patients with recurrent glioblastoma (GBM). Methods: Patients with recurrent GBM operated on between 2007 and 2014 were divided into 3 groups: >65 years with BCNU wafer implantation, >65 years without BCNU wafer implantation, and ≤ 65 years with BCNU wafer implantation. We compared survival and complications. Results: A total of 79 patients were identified: 24 in the older BCNU group (median age 68.2 years, 33.3% with a methylated MGMT promoter), 16 in the older non-BCNU group (median age 68.6 years, 31.3% with a methylated MGMT promoter), and 39 in the younger BCNU group (median age 56.8 years). Survival after progression was 9.2 months in the elderly BCNU group and 7.6 months in the elderly non-BCNU group ( p = 0.34); overall survival was 17.2 and 15.9 months, respectively ( p = 0.35). We found a tendency toward a higher rate of seizures and pneumonia in the older BCNU group. Conclusion: BCNU wafer implantation after resection of recurrent GBM is a reasonably safe treatment in patients aged >65 years. Seizures and systemic infections may occur more frequently, but the trade-off is still favorable as survival may be influenced positively. Higher age should not be regarded as a contraindication for BCNU wafers.