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 DNA repair protein 06-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine in DNA and thus may protect cells against genotoxic effects of agents inducing this lesion. To analyze quantitatively the level of protection mediated by MGMT against antineoplastic drugs, we determined the cytotoxic and recombinogenic (sister-chromatid exchange inducing) effects of various chemotherapeutic agents in a pair of isogenic Chinese hamster cell lines deficient and proficient for MGMT, generated upon transfection with human MGMT cDNA. Furthermore, we compared the responses of the human cell lines Hela MR (MGMT deficient) and HeLa S3 (MGMT proficient) t o the various agents.It is shown that: (I) MGMT proficient cells are resistant in cell killing to the methylating drug streptozotocin and all the chloroethylating nitrosoureas tested. There was a marked agent specificity in protection. The level of resistance provoked by MGMT increased in the order BCNU < CCNU < ACNU < HeCNU < streptozotocin. (2) MGMT did not protect cells against killing induced by chlorambucil, cisplatin, melphalan, activated cyclophospharnide (mafosfamide) and activated ifosfamide (4-hydroperoxy-ifosfamide). (3) MGMT caused protection against the recombinogenic effect of all nitrosoureas tested. The lowest level of protection was again observed for BCNU, followed by CCNU, ACNU < HeCNU < streptozotocin. (4) MGMT proficient cells did not exhibit resistance in SCE induction towards cyclophosphamide (activated by microsornes), 4-hydroperoxy-ifosfamide, mafosfamide, chlorambucil and melphalan. Some protection was afforded, however, against cisplatin (and transplatin). This effect was abolished by pretreatment of cells with 06-benzylguanine, which depletes MGMT, indicating that some lesion($ induced by cisplatin giving rise to SCEs can be repaired by MGMT. Taken together, these results indicate that streptozotocin, HeCNU and ACNU are more selective than CCNU and BCNU in killing MGMT deficient cells, and that in the cases of cyclophosphamide, ifosfamide, chlorambucil, cisplatin and melphalan MGMT is not involved in mediating cytotoxic drug resistance.o 1996 Wiley-Liss, Inc.

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of < 40 kDa. As for other bacterial PLCs, the enzyme requires Ca 21 and Zn 21 for activity; dithiothreitol affected the activity due to its chelation of Zn 21 , but this inhibition could be compensated for by addition of ZnCl 2 . The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at < 420 mM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH 2 -terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis.

The targeting of mRNA with antisense oligonucleotides is increasingly employed to inhibit the expression of gene function. Since the level of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is decisive in protection of cells against damage produced by alkylating agents, including cytostatic drugs, the targeted inhibition of this repair activity might be of importance for therapeutic approaches. In order to investigate whether antisense targeted MGMT depletion is feasible to transiently modify the sensitivity of cells to anticancer drugs, we studied the expression of MGMT and cellular sensitivity upon inhibitor and antisense treatment using CHO transfectants expressing human MGMT. It was shown by polymerase chain reaction that antisense oligonucleotides specifically inhibited MGMT mRNA level. Nevertheless, MGMT protein was found not to be reduced significantly, as demonstrated by Western blotting. Correspondingly, no significant decrease in MGMT activity was observed, as measured 36 h after MGMT antisense oligonucleotide administration. Given together with the MGMT depleting agent O6-methylguanine, reduction in MGMT protein as well as activity was found. MGMT antisense oligonucleotide enhanced the sensitivity of cells to the tumor therapeutic drug mitozolomide, as measured by sister chromatid exchange formation. This sensitization was further enhanced by combined treatment with antisense oligonucleotide and O6-methylguanine, indicating that MGMT antisense can be supportive in sensitization of cells to an alkylating drug.