Effect of cell confluence on ultraviolet light apoptotic responses in DNA repair deficient cells
Introduction
Ultraviolet light (UV) radiation is a constant source of potentially deleterious effects on sun exposed areas of the body. These deleterious effects are due mainly to DNA damage. The main lesions caused by UV on DNA are cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts ((6-4) PPs), which are normally removed by an efficient nucleotide excision DNA repair system (NER) [1], [2], [3]. Several proteins with distinct activities are involved in this repair mechanism such as XP (XPA through XPG) and CS proteins (CSA and CSB). XP proteins are mutated in xeroderma pigmentosum, a rare disease characterized by photosensitivity, high incidence of skin cancer and sometimes associated to neurological symptoms [4]. CS proteins are mutated in Cockayne’s syndrome, characterized by growth and mental retardation, retinal abnormalities and severe photosensitivity [5]. Mutations in XP genes can also give rise to another disease called trichothiodystrophy (TTD), characterized by brittle hair and nails due to deficiency in sulfur-rich protein synthesis. TTD result from mutations in either XPB or XPD genes. Mutations in TTDA gene are also responsible for a TTD phenotype [6].
The NER system works in two pathways: repairing the transcribed strand of active genes (transcription-coupled repair—TCR), or repairing the rest of the genome (global-genome repair—GGR) [3]. Cells from XP patients are deficient in both TCR and GGR, with the exception of the XPC complementation group, which is exclusively impaired in GGR. On the other hand, CS cells show exclusive impairment in TCR [5].
Upon UV irradiation, cells stall the cell cycle, generally by p53 activation [7], [8], to allow the NER system enough time to correct DNA mutations. If damage is too severe, the cell will trigger a sophisticated apoptosis-inducing mechanism that prevents it from passing on mutations if it divides. Apoptosis is a controlled mode of cell death, characterized by cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentation in a characteristic pattern [9], [10]. Apoptosis induction can occur as a consequence of a series of extracellular stimuli, amongst which, reactive oxygen species, ionizing/UV radiation, chemotherapy drugs, cytokines, growth factor withdrawal, etc. [11]. During the final steps of this process, cell proteins are degraded by cysteine proteases, called caspases, which causes the typical morphology mentioned above.
In mammalian cells, apoptosis induced by UV light has been shown to be triggered mainly by CPD photoproducts [12], [13]. In this work, we examined UV-induced apoptosis using HeLa cells expressing a CPD-photolyase gene. UV-induced inhibition of RNA synthesis and apoptosis were greatly prevented by heterologous photorepair in these cells, suggesting that RNA polymerases II complexes blocked by CPDs signal to programmed cell death. In order to better understand the mechanisms involved in apoptosis induced by UV light, we have also studied the influence of the cell cycle on apoptotic responses of NER deficient cells after UV injury. For this, we have used cells from xeroderma pigmentosum patients, which are notably more sensitive to apoptosis by UV radiation [14], [15]: XPA and TTD/XPD, deficient for GGR and TCR, as well as XPC (GGR deficient) and CSB (TCR deficient) primary skin fibroblasts. Here we confirm that XPA cells are more sensitive to UV radiation when compared to XPC cells, as has been previously shown [15]. However, confluent XPC and TTD/XPD cells were much more resistant to apoptosis induction by UV when compared to non-confluent cells, while confluent XPA cells showed a similar sensitivity to low doses of UV when compared to non-confluent cells. Confluence conditions in cell culture may interfere in replication and transcription complexes stalled by CPD lesions, thus modifying the apoptotic response observed in confluent primary cells. Additionally, this response is dependent on the NER status, indicating the participation of DNA repair enzymes in DNA damage-induced apoptosis. This is the first report to show the effect of cell confluence on UV-induced apoptosis in DNA repair-deficient cells.
Section snippets
Reagents
Dulbecco’s modified Eagle’s medium, and the penicillin/streptomycin solution were from Life Technologies Inc. (Grand Island, NY). Fetal calf serum was from Cultilab (Campinas, Brazil). Propidium iodide was from Sigma (St. Louis, MO). All other reagents were of analytical grade.
Cell lines and culture
We used primary fibroblasts derived from skin biopsies from xeroderma pigmentosum patients (complementation groups A e C—XPA and XPC—XP456VI and XP016VI cells, respectively), from Cockayne’s syndrome patients
Recovery of UV-induced RNA synthesis inhibition by CPD-photolyase in HeLa cells
Cyclobutane pyrimidine dimers are the most frequent photoproducts generated in DNA after UV irradiation [1], [19] and induce several metabolic responses, such as cell cycle arrest, RNA transcription inhibition and apoptosis [20]. In order to investigate the role of these lesions in UV-induced apoptosis in human cells, we generated HeLa cells expressing a marsupial CPD-photolyase gene (gene phr [12]). Flow cytometry analysis was performed on HeLa parental and phr-expressing cells to quantify
Discussion
Previous results demonstrated that CPD photoproducts are the initial signals that trigger apoptosis in human cells after UV irradiation [12]. The presence of these lesions in the transcribed strand of active genes is supposed to be responsible for programmed cell death in response to DNA damage, since TCR deficient cells display a higher level of UV-induced apoptosis than repair proficient cells after low UV dose exposure [22]. Although transcriptional arrest may be only a passive consequence
Acknowledgements
The authors would like to thank Dr. B. Kaina (Mainz, Germany) for critical comments and helpful discussions. This work is supported by FAPESP (São Paulo, Brazil) and CNPq (Brası́lia, Brazil). H. Carvalho, R.M.A. Costa, V. Chiganças, R. Weinlich and G. Brumatti acknowledge their fellowships from FAPESP.
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Present address: Departamento de Análises Clı́nicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, Brazil.