The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of tumor cells to the cytostatic activity of chemotherapeutic alkylating agents (methylating and chloroethylating nitrosoureas) and is effective in protecting normal cells against genotoxic and carcinogenic effects resulting from DNA alkylation. Therefore, the level of expression of MGMT is significance for the response of both the tumor and the non-target tissue following application of nitrosoureas in tumor therapy. To determine the expression of MGMT in tumor tissue, we have assayed MGMT activity in 68 breast carcinomas and 38 brain tumors. There was a wide variation of MGMT expression in breast carcinomas ranging from below the level of detection up to 863 fmol/mg protein. About 4% of breast tumors did not display detectable MGMT, 15% had activity lower than 100 fmol/mg protein, and 26% expressed more than 500 fmol/mg. The mean level of expression was 321 fmol/mg. In brain tumors (astrocytoma WHO grade I, II, and III, and glioblastoma WHO grade IV) the MGMT activity was generally lower than in breast tumors, ranging from below the level of detection up to 238 fmol/mg. The mean level of expression was 55 fmol/mg. Five percent of the brain tumors had no detectable MGMT activity. The MGMT repair activity correlated well with the amount of MGMT protein present in tumor samples, as shown by Western-blot analysis, indicating that loss of MGMT repair activity is due to inability of these tumor cells to synthesize the protein.
The proto-oncogene c-fos encodes a nuclear protein that forms together with c-Jun or other members of the Jun family the transcription factor AP-1. The c-fos gene is inducible by UV radiation and other DNA damaging treatments which may indicate that it is required in defence against DNA damaging agents. To address this hypothesized function of c-Fos, we have compared the response of mouse fibroblasts deficient in c-Fos with the corresponding wild-type cells towards the genotoxicity of UV radiation. It is shown here that lack of c-Fos renders cells hypersensitive to the cytotoxic effect of UV light and gives rise to significant increases of UV-induced chromosomal mutations and DNA breakage. Cells lacking c-Fos were basically able to perform UV-induced repair replication, as measured by unscheduled DNA synthesis. However, with high doses of UV c-Fos deficient cells proved to be less efficient in repair synthesis than wild-type cells. Measurement of overall DNA synthesis after UV irradiation revealed that cells deficient in c-Fos are more inhibited in their recovery from the UV-induced block to replication. These data strongly suggest that c-Fos is involved in regulating the timing of DNA replication after UV irradiation by abolition of the UV-induced block to replication and thus appears to play a decisive role in the cellular defence against the genotoxic effects induced by UV radiation.
The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a
TRAC
locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding
TRAC
locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale.
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