Inhibition of MT-450 rat mammary tumour growth by antibodies recognising subtypes of blood group antigen B.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2.

Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex-, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models.

O6-Methylguanine (O6-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O6-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation.

Overexpression of N-methylpurine-DNA glycosylase in Chinese hamster ovary cells renders them more sensitive to the production of chromosomal aberrations by methylating agents--a case of imbalanced DNA repair.

The main N-alkylation products induced in DNA by methylating mutagens (7-methylguanine, 3-methyladenine, 3-methylguanine) are removed by excision repair involving, in the first step of the repair pathway, N-methylpurine-DNA glycosylase (MPG). To elucidate the significance of excision repair of N-alkylpurines in the defense of cells against alkylating agents we have modulated the efficiency of removal of N-methylpurines in Chinese hamster cells by transfecting them with the human MPG cDNA cloned into a mammalian expression vector. Although the stably transfected cells had a significantly higher capacity for removal of N-methylpurines from DNA, they did not gain protection against the cytotoxic and mutagenic effect of alkylating agents.

Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges, chromosomal aberrations, and gene mutations: new insights gained from studies of genetically engineered mammalian cell lines.

O6-methyl- and O6-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkylating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O6-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O6-methylguanine-DNA methyltransferase (MGMT).

Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells.

N-Alkylpurines induced in DNA by simple monofunctional alkylating agents are known to be cytotoxic and possibly indirectly mutagenic. These adducts are removed by the ubiquitous N-methylpurine-DNA glycosylase (MPG) in a multistep repair pathway. Chinese hamster ovary (CHO) cell clones expressing 2- to 16-fold enhanced levels of MPG activity were isolated from cells stably transfected with human MPG cDNA expression plasmids.

Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU).

Identification of human genes involved in repair and tolerance of DNA damage.

An overview is presented on strategies of cloning mammalian DNA repair genes. Complementation of human and rodent repair defects and mutagen hypersensitivities by chromosome and DNA mediated gene transfer and mRNA microinjection is described, and the features of the cloned human DNA repair genes are summarized. It is shown that transfection of repair deficient cell lines with cloned bacterial and human genes may give rise to protection from the genotoxic effects of mutagens.

Lymphocytes infected with Theileria parva require both cell-cell contact and growth factor to proliferate.

Lymphocytes infected with the intracellular parasite Theileria parva proliferate continuously as lymphoblastoid cell lines. We have previously shown that the continuous proliferation of the T. parva-infected (Tpi) cell line TpM(803) is mediated in part by an autocrine mechanism (Dobbelaere, D.

Expression and characterization of the interleukin 2 receptor in Theileria parva-infected bovine lymphocytes.

We have previously shown that interleukin 2 receptors (IL2R) are constitutively expressed on the surface of bovine lymphocytes infected with the parasite Theileria parva (Dobbelaere, D.A.E.

Theileria parva infection induces autocrine growth of bovine lymphocytes.

Bovine lymphocytes infected with the parasite Theileria parva continuously secrete a growth factor that is essential for their proliferation in vitro and also constitutively express interleukin 2 receptors on their surface. Dilution of the secreted growth factor, caused by culturing cells at low density, results in retardation of culture growth. Human recombinant interleukin 2, however, effectively substitutes for the diluted growth factor by restoring normal growth rates and also allows Theileria-infected cells to be grown at low density without the use of feeder layers.

Rejoining of double strand breaks in normal human and ataxia-telangiectasia fibroblasts after exposure to 60Co gamma-rays, 241Am alpha-particles or bleomycin.

The rejoining of DNA double strand breaks (dsb) induced by 60Co gamma-rays, 241Am alpha-particles or bleomycin was measured by neutral filter elution. In agreement with their colony-forming ability, ataxia-telangiectasia cells (AT2BE) and normal fibroblasts exhibited similar dsb rejoining capacity following alpha-irradiation, but showed marked differences in the rejoining kinetics of dsb induced by gamma-rays or bleomycin.

Relationship between double strand break rejoining and G2 block formation in V79 cells.

Caffeine and hypertonicity affect the survival of gamma-irradiated Chinese Hamster V79 cells in different ways: while caffeine reduces the shoulder of the dose effect curve, hypertonic treatment mainly affects its final slope suggesting that different types of damage and (or) repair mechanisms are involved. Rejoining of DNA double strand breaks (dsb), as measured by neutral filter elution technique, exhibits a fast and a slow component, indicating that dsb rejoining consists of two different processes. Hypertonicity causes a temporary inhibition of the fast rejoining step but has no effect on the overall rejoining efficiency.

Radiation induced DNA double strand breaks are rejoined by ligation and recombination processes.

Using the method of filter elution of double stranded DNA under neutral conditions we have shown that most of gamma-ray induced double strand breaks (DSB) are rejoined in both mammalian and bacterial cells. Rejoining also occurs in the G1 phase in V79 Chinese hamster cells and under different growth conditions. Within 8 minutes at 37 C, half the breaks are rejoined.

Rejoining of DNA double-strand breaks in human fibroblasts and its impairment in one ataxia telangiectasia and two Fanconi strains.

Using the technique of neutral elution through polycarbonate filters as a measure of DNA length, and hence of the number of double-strand breaks incurred as a result of radiation damage, we found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C). This fast rejoining was impaired in fibroblasts from one patient with Ataxia telangiectasia and in fibroblasts from two patients with Fanconi's anemia. Also the number of residual breaks after several hours of repair was higher than in control cells.

Negative pion irradiation of mammalian cells. III. A comparative analysis of DNA strand breakage, repair and cell survival after exposure to pi-mesons and X-rays.

The yields of immediate and residual breaks in DNA of X- and peak pion irradiated Chinese hamster cells were measured by hydroxylapatite chromatography in order to investigate the r.b.e.

Properties of an endonuclease activity in Micrococcus luteus acting on gamma-irradiated DNA and on apurinic DNA.

A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities.

End group characterization in DNA of thymocytes after low doses of ionizing radiation.

Investigations into the configuration of the radiation induced strand breaks in the low dose range are presented. DNA sections containing the radiation induced strand breaks were separated from the undamaged sections in order to increase the concentration of lesions. The configuration of 3'terminals in damaged DNA from gamma-irradiated thymocytes was analysed studying the priming ability for the DNA polymerase I.

Oxygen--effect on strand breaks and specific end-groups in DNA of irradiated thymocytes.

Thymocytes were irradiated with fast electrons up to 6 Mrad in the presence and absence of oxygen. The cells were treated before irradiation with a cold shock to prevent any repair rejection during irradiation. The DNA isolated subsequently was analysed for double-strand breaks (dsb), actual single-strand breaks (ssb) and alkali-induced strand breaks (alisb).