RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae. (1/4844)

The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53.  (+info)

Genome reduction in a hemiclonal frog Rana esculenta from radioactively contaminated areas. (2/4844)

A decrease in genome size was found in the hemiclonal hybridogenetic frog Rana esculenta (R. ridibunda x R. lessonae) from areas of radioactive contamination that resulted from the Chernobyl fallout. This genome reduction was of up to 4% and correlated with the background level of gamma-radiation (linear regression corresponded on average to -0.4% per doubling of radiation level). No change in genome size was observed in the coexisting parental species R. lessonae. There was no correlation between genome size and body mass in R. esculenta froglets, which have metamorphosed in the year of the study. The hemiclonal forms may become a suitable object for study on biological significance of individual DNA sequences (and of genome size as a whole) because mutant animals with deletions in a specified genome can arise after a low radiation dose. The proneness to genetic damage makes such forms also a prospective bioindicator of radioactive (and possibly other mutagenic) pollution with the effects of genetic damage conveniently and rapidly monitored by DNA flow cytometry.  (+info)

Loss of normal G1 checkpoint control is an early step in carcinogenesis, independent of p53 status. (3/4844)

Recent studies have described a diminished radiation-induced G1 arrest in some wild-type (wt) p53 human tumor cell lines compared to normal human fibroblasts. However, the significance of this finding was unclear, particularly because tumor cell lines may have accumulated additional genetic changes after long periods in culture. Because malignant transformation of individual cells is thought to be an early step in carcinogenesis, we have used a model system of normal and transformed mouse fibroblast 10T1/2 cell clones to examine whether loss of G1 checkpoint control may be an early event in tumor development and to study the relationships between G1 arrest, radiosensitivity, and genetic alterations. Twelve transformed clones were established from type III foci induced by irradiation of normal 10T1/2 cells and were compared with six clones derived from wt 10T1/2 cells. Three of the transformed clones expressed mutant p53; two of these had the same point mutation at codon 132 (exon 5), and one had a point mutation at codon 135. The remaining transformed and normal clones had wt p53 status. The radiosensitivity of transformed clones, as measured by a clonogenic assay, was similar to that of normal clones; the three clones with mutant p53 did not differ from the others. There was no relationship between G1 arrest and radiosensitivity. Normal 10T1/2 cell clones showed a transient G1 arrest lasting approximately 9 h after 6 Gy of irradiation. This G1 arrest was either absent or markedly reduced in all of the transformed clones, regardless of p53 status. These results suggest that diminished G1 checkpoint control is an early event in the process of carcinogenesis that is associated with the malignant transformation of individual cells and is independent of p53 status.  (+info)

Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant. (4/4844)

The xeroderma pigmentosum (XP) variant (XPV) is a form of XP that has normal excision repair but shows defective DNA replication after UV irradiation. In developing various transformed fibroblast cell lines from these patients, we have found that there are significant phenotypic changes in transformed cells that seem to correlate with inactivation of p53. After transformation with SV40, XPV cell lines are only slightly UV sensitive, like their primary counterparts, but their sensitization with caffeine and the induction of sister chromatid exchanges (SCEs) by UV irradiation are greatly enhanced. After transformation by HPV16 E7, which targets the retinoblastoma cell cycle regulatory gene, there is no change in the UV sensitivity of XPV cells; but, when transformed by HPV16 E6 or E6 and E7 combined, there is a large increase in UV sensitivity and in the induction of SCEs. These changes are not associated with any detectable changes in the reactivation of an externally irradiated luciferase expression vector, the excision of cyclobutane pyrimidine dimers from bulk DNA, or unscheduled DNA synthesis and, therefore, do not involve excision repair. We suggest that if SCEs represent homologous recombination between sister chromatids, then in the absence of p53 function, the DNA chain arrest typical of UV-damaged XPV cells initiates strand exchange during recovery. In untransformed cells with normal p53, the preferred mode of recovery would then be replication bypass. The symptoms of elevated solar carcinogenesis in XPV patients may, therefore, be associated with increased genomic instability in cells of the skin in which p53 is inactivated by UV-induced mutations.  (+info)

Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs. (5/4844)

Tumor vaccines and gene therapy have received significant attention as means of increasing cellular and humoral immune responses to cancer. We conducted a pilot study of seven research dogs to determine whether intradermal injection of canine tumor cells transfected via the Accell particle-mediated gene transfer device with the cDNA for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) would generate biologically relevant levels of protein and result in demonstrable histological changes at sites of vaccination. Tumor cell vaccines of 10(7) irradiated canine melanoma cells were nontoxic, safe, and well tolerated. No significant alterations in blood chemistry values or hematological profiles were detected. A histological review of control vaccine sites revealed inflammatory responses predominated by eosinophils, whereas vaccine sites with hGM-CSF-transfected tumor cells had an influx of neutrophils and macrophages. Enzyme-linked immunosorbent assays of skin biopsies from vaccine sites had local hGM-CSF production (8.68-16.82 ng/site of injection) at 24 hours after injection and detectable levels (0.014-0.081 ng/site) for < or =2 weeks following vaccination. Flow cytometric analysis of hGM-CSF-transfected cells demonstrated < or =25% transfection efficiency, and hGM-CSF levels obtained during time-course assays demonstrated biologically relevant levels for both irradiated and nonirradiated samples. These data demonstrate the in vivo biological activity of irradiated hGM-CSF-transfected canine tumor cells and help provide evidence for a valid translational research model of spontaneous tumors.  (+info)

Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema. (6/4844)

We determined the effects of low dose radiation (<200 cGy) on the cell-cell integrity of confluent monolayers of pulmonary microvascular endothelial cells (PMEC). We observed dose- and time-dependent reversible radiation induced injuries to PMEC monolayers characterized by retraction (loss of cell-cell contact) mediated by cytoskeletal F-actin reorganization. Radiation induced reorganization of F-actin microfilament stress fibers was observed > or =30 minutes post irradiation and correlated positively with loss of cell-cell integrity. Cells of irradiated monolayers recovered to form contact inhibited monolayers > or =24 hours post irradiation; concomitantly, the depolymerized microfilaments organized to their pre-irradiated state as microfilament stress fibers arrayed parallel to the boundaries of adjacent contact-inhibited cells. Previous studies by other investigators have measured slight but significant increases in mouse lung wet weight >1 day post thoracic or whole body radiation (> or =500 cGy). Little or no data is available concerning time intervals <1 day post irradiation, possibly because of the presumption that edema is mediated, at least in part, by endothelial cell death or irreversible loss of barrier permeability functions which may only arise 1 day post irradiation. However, our in vitro data suggest that loss of endothelial barrier function may occur rapidly and at low dose levels (< or =200 cGy). Therefore, we determined radiation effects on lung wet weight and observed significant increases in wet weight (standardized per dry weight or per mouse weight) in < or =5 hours post thoracic exposure to 50 200 cGy x-radiation. We suggest that a single fraction of radiation even at low dose levels used in radiotherapy, may induce pulmonary edema by a reversible loss of endothelial cell-cell integrity and permeability barrier function.  (+info)

In situ repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in human skin exposed to solar simulating radiation. (7/4844)

DNA repair is crucial to the integrity of the human genome. The ultraviolet radiation portion of solar radiation is responsible for the rising incidence of skin cancer, one of the most common types of cancer in humans. We applied a recently developed 32P-postlabeling technique to measure the in situ DNA repair efficiency of solar-simulated radiation induced cyclobutane pyrimidine dimers and 6-4 photoproducts in the skin of nine healthy volunteers with skin type II. Our results show about 6-fold interindividual variations in the level of DNA damage after exposure to an equal biologic dose - 2 minimal erythema doses. The kinetics of DNA repair indicated a base sequence dependence of the repair process. The DNA repair efficiency showed a 20-fold difference in volunteers. An age-related decrease of DNA repair capacity was observed; however, the data are limited due to a small number of subjects and a narrow age range. The variable response in DNA damage levels and individual differences in DNA repair efficiency suggest a susceptible subgroup of people probably with a higher skin cancer risk.  (+info)

Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation? (8/4844)

Progression of BY-2 tobacco cells through the cell cycle was followed after treatments with ultra violet (UV) and salicylic acid (SA) used as a potent inhibitor of the octadecanoid pathway which can mediate response to UV irradiation. Cells in S phase were more sensitive than G0/G1 or G2 cells to UV irradiation. Although SA efficiently blocked cells in G0/G1 or G2, it did not block S phase synchronized cells. UV and SA applied simultaneously to cells in G0/G1 delayed the cell cycle progression more than each one separately. Therefore UV irradiation and SA act as agonists to arrest BY-2 cells at cell cycle entry. To further investigate the signalling pathway mediating UV response, we complemented a UV-sensitive Escherichia coli strain with a Nicotiana xanthi cDNA expression library. A cDNA (arcA3) whose coding sequence is identical to the 2,4-D induced arcA cDNA cloned by Ishida et al. (1993) was isolated. We show that arcA3 transcription is induced at cell cycle entry but not directly by the 2,4-D treatment. Moreover, arcA3 transcription is induced prior to the restriction point as shown with the CDK inhibitor roscovitine. The arcA3 transcription level is increased by UV irradiation but prevented by SA. Indeed, addition of SA prior to UV irradiation blocks the induction of arcA3 transcription. This suggests that arcA3 gene is modulated in both UV and SA responses, the SA effect preceding the UV step. Since arcA3 is 67% similar to RACK1 (functional homology), a rat intracellular receptor for protein kinase C, and possesses identical PKC fixation motifs, it is hypothesised that the arcA3 gene is involved in UV and SA cell cycle arrest.  (+info)