Objective
Research has been carried out to develop an assay system for the assessment of the contribution of ionizing and ultraviolet (UV) radiation to carcinogenesis and consisting of primary or secondary cell cultures from mice transgenic for an activated oncogene. None of the cells used were transformed upon radiation and further work will be carried out to create transgenic mice expressing selected oncogenes specifically in the skin for use as a model for UV carcinogenesis.
Exposure of human cells to UV or ionizing radiation results in induction of stress phenomena, including enhanced reactivation (ER) of UV treated virus and enhanced mutagenesis (EM) of untreated virus. It was noted that the ER response was absent in Xeroderma pigmentosum (XP) patients who also lacked tumors in sunlight exposed skin, suggesting the response might be coregulated with oncogene activation. Since EM was unchanged in ER(-) patients, the ability to induce point mutations in response to deoxyribonucleic acid (DNA) damage was not affected. Cells from UV sensitive Trichothiodystrophy patients, also non cancer prone, showed no ER response but cells from hereditary cancer prone syndromes responed to UV irradiation with superinduction of ER. Induction by UV irradiation of UV inducible genes was not affected in ER(-) and ER(super+) cells, but an abnormality was found in UV induced stabilization of the tumour associated protein p53. In ER(-) cells no UV induced stabilization of p53 was found but it was discovered that p53 was already constitutively stabilized in such cells.
The mutagenic properties of an abasic site located opposite a guanine residue were studied in cultured mammalian cells by insertion into shuttle vector able to replicated in simian cells and in bacteria. Plasmid deoxyribonucleic acid (DNA) was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Opposite a guanine, the abasic site was repaired error free or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at - frequency of 1% in control host cells and at more than 3% in ultraviolet (UV) preirradiated host cells. No preferential insertion of a particular base was found opposite the abasic site.
Epstein-Barr virus (EBV) based shuttle vectors have been developed for gene amplification studies. The vectors carried a target gene for mutagenesis study (lacZ'), which allowed accurate monitoring of the amplification process.
Oncogene activation has been studied in skin tumours from Xeroderma pigmentosum (XP) and non-XP patients. Genomic DNA from XP tumours was found to have high levels of Ha-ras gene amplification not present in skin tumours from normal patients. Screening of ras mutations in skin tumours detected mutations in 55% of XP tumours compared to 20% of controls, ie unrepaired DNA lesions persisting in XP skin results in higher levels of ras mutations than in carcinomas from patients with normal repair capacities. The high level of oncogene amplification in XP cells is consistent with blockage of DNA polymerase at unrepaired lesions. The presence of activated and mutated oncogenes in the same cell explains the high cancer frequency in XP patients.
Studies have been carried out into the isolation of mammalian cell protein similar to the bacterial recA protein, which is important in radioprotection. The results were as follows:
demonstration that in mammalian species (mouse and man) there are some proteins sharing antigenic determinants with Escherichia coli recA protein;
isolation of a mouse complementary deoxyribonucleic acid (cDNA) of 601 nucleotides that codes for a polypeptide expressing the strongest immunoreactive recA epitope;
cloning of a complete mouse cDNA of 1413 nucleotides called KIN17 cDNA, encoding a 44 kD protein;
determination of the kin17 amino acid sequences having a zinc finger motif, nuclear localization of KIN17 messenger ribonucleic acid (mRNA) of 1.8 kb, which is expressed in transformed mouse neuroendocrine AtT-20 cells at a high level;
localization by cytogenetic mapping indicating that the KIN17 gene is located in band A of mouse chromosome 2;
demonstration that genomic sequences homologous to KIN17 are present in human DNA.
Research has been carried out into the nature of the signal triggering the inducible enhanced reactivation (ER) recovery process and into the radiation induced disturbances of gene expression in human cells.
The mechanism of ER induction in human cells was studied by investigation of whether the factor secreted by irradiated cells was able to trigger ER of single stranded or double stranded deoxyribonucleic acid (DNA) viruses (H-1 and HSV-1, respectively) in unirradiated cells. It was found that H-1 reactivation was not enhanced but HSV-1 reactivation was enhanced. However, the ER of HSV-1 was lower than that obtained after direct irradiation of the cells. The signal produced by extracellular factors may partially activate ER of HSV-1 but may be too low to stimulate H-1 reactivation.
Post transcriptional effects of radiation have been investigated using exogenous agents which turn on production of messenger ribonucleic acid (mRNA). It was found that steady state levels of cytokine and poly(I)-poly(C) induced transcripts were higher in irradiated cells and that the stimulation was dose dependent.
Chromosome analysis has been carried out in cultured fibroblasts of skin from Xeroderma pigmentosum (XP) patients and their families and anomalies found were balanced translocations, deletions and inversions. Investigations in Trichothiadystrophy (TTD) patients showed an association at the genetic level between TTD and patients carrying the XP-D mutations.
Studies were carried out into the role of poly(adenosine diphosphate-ribose)polymerase (pADPRP), which is activated in response to deoxyribonucleic acid (DNA) damage, during proliferating activity of mammalian cells in rat liver regeneration. Messenger ribonucleic acid (mRNA) levels of the enzyme had a peak early on, before onset of DNA synthesis, and a second peak which reached a maximum after the peak of DNA synthesis. A complementary DNA (cDNA) probe for the enzyme showed the transcription rate increased during all proliferation preceding the increase in DNA synthesis. The early increase probably resulted from activation of preexisting pADPRP molecules, the second phase being associated with de novo synthesis of the protein, showing involvement of pADPRP in earlyand late events of cell proliferation. Investigation of pADPRP inhibitors, based on 3-aminobenzamide, showed that the most powerful inhibitors were characterized by substitution in position 3 of the amidic function and were devoid of cellular toxicity.
Chinese hamster ovary (CHO) cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) were selected by growing them on 2-amino-6-mecaptopurine. The cell line was used for the introduction, propagation and maintenance of selected genes in mammalian cells. Thus, after cotransfection with pSV2-gpt vector and piH3-CD2 vector using the calcium phosphate coprecipitation technique, surviving cells were grown selectivity in HAT medium. Recombinant deoxyribonucleic acids (DNA) containing pSV2-gpt as a selective marker may be useful for cotransformation of nonselectable genes. The effect of low doses of radiation will be studied on expression of the CD2 gene.
Current risk estimates for the effects of radiation are based on the assumption of linear dose response relationships. Whether the assumption is justified is a question of major importance for public health considerations. This new proposal aims to explore the existence and mechanisms of response modification. The term response modification should include all processes that are induced by radiation, and that influence the subsequent fate of the cell and organism (other operational terms for the same processes are UV-response and stress response.
This programme consists of a collaboration between a number of European laboratories that have made several contributions to the study of stress responses in bacterial and mammalian cells. Devoret is one of the pioneers in bacterial SOS genetics, a system that serves as a model for the mammalian stress response. Rommelaere, Sarasin and Van der Eb were among the first to detect the stress responses of Enhanced Reactivation and Enhanced Mutagenesis in mammalian cells, whereas Bertazzoni has joined the field of radiation-induced responses more recently, concentrating primarily on hereditary disorders characterized by chromosomal fragility and tumour proneness.
In the present proposal, Devoret's group is focusing on the characterization of a recA-like protein in mammalian cells. In E.coli the recA protein plays a key role in protecting the genetic material against deleterious effects of radiation, and it seems likely that the mammalian homolog of this protein has a comparable function. The groups of Rommelaere and Van der Eb are investigating the phenomenon of radiation-induced Enhanced Reactivation (ER). Within this context, Rommelaere is concentrating primarily on the mechanism of induction of ER, whereas Van der Eb is studying abnormal ER responses in cells from certain cancer prone genetic diseases. In the same framework Sarasin's groups is developing systems to quantify radiation-induced gene amplification and to study the underlying mechanism of this phenomenon.
Finally, the group of Bertazzoni is investigating spontaneous and radiation-induced chromosome fragility phenomena, as well as the role of poly(ADP) ribosylation in the cellular response to radiation damage. Several of the groups of this joint research programme are collaborating with P. Herrlich's and P. van de Putte's laboratories, which have made important contributions to the discovery of UV-inducibility of genes and the isolation of new classes of UV-inducible genes. Collaboration also exists with J.W.I.M. Simons' laboratory on mutagenesis in mammalian cells.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- medical and health sciences health sciences public health
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences genetics mutation
- medical and health sciences clinical medicine oncology
- medical and health sciences health sciences infectious diseases DNA viruses
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Coordinator
2333 AL LEIDEN
Netherlands
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