Flow cytometric analysis of DNA sensitivity to nuclease S1 in UVC-irradiated human fibroblasts

The sensitivity of human fibroblast DNA towards the activity of nuclease S1 was investigated in situ after irradiation of the cells with UVC light. DNA digestion was assessed using flow cytometry by staining the nuclease-resistant DNA fraction with the specific fluorochrome propidium iodide (PI). The results showed a non-linear dependence of DNA digestion on the UVC irradiation dose between 0 and 20 J m-2. About 70% of the UVC-induced DNA sensitivity to nuclease S1 was lost after 30 min of repair. These results suggest that flow cytometry may be useful for assessing the heterogeneity of cell response to UV damage

Nuclear association of cyclin D1 in human fibroblasts: tight binding to nuclear structures and modulation by protein kinase inhibitors.

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway

Nuclear binding of cell cycle-related proteins: Cyclin A versus proliferating cell nuclear antigen (PCNA).

We have investigated the cell cycle-dependent nuclear binding of cyclin A and of the proliferating cell nuclear antigen (PCNA) in asynchronously growing human fibroblasts. To this purpose, we have applied flow cytometry immunofluorescence, a powerful technique for elucidating the cell cycle phase during which the nuclear binding occurs. We have observed that, in striking contrast with the distribution of nuclear-bound PCNA which is restricted to S phase, the immunofluorescence signal of the nuclear-bound form of cyclin A is high in the G1 and G2 phases of the cell cycle. These results suggest the involvement of nuclear-bound cyclin A in the G1/S and G2/M phase transitions

Involvement of proliferating cell nuclear antigen in DNA repair after damage induced by genotoxic agents in human fibroblasts

The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites during the process of DNA repair, was investigated in human diploid fibroblasts after treatment with different genotoxic agents. For this purpose, confluent cultures were treated with agents that form primarily DNA adducts, such as u.v.-C, 8-methoxypsoralen (8-MOP) and 4,4',6-trimethylangelicin (TMA), or with agents that induce strand breaks, such as X-rays or bleomycin (BLM). Chromatin-associated PCNA was detected with a monoclonal antibody and indirect immunofluorescence labelling. Quantitative analysis was performed by flow cytometry. Not all of the tested agents were able to activate the association of PCNA with chromatin. X-rays, u.v.-C and BLM induced a significant increase in PCNA complex as compared to the control samples. In contrast, 8-MOP and TMA did not show any detectable effect on the levels of associated PCNA, even at post-incubation repair times as long as 10 h. However, these drugs damaged DNA, as shown by the formation of micronucleated cells 48 h after treatment. The lack of PCNA activation was not due to an inhibition of the repair mechanism, since in TMA-treated fibroblasts, subsequent irradiation with u.v.-C induced an increase in PCNA levels comparable to that found in cells treated with u.v.-C alone. These results indicate that PCNA is involved in DNA excision repair of genotoxic agents, but suggest that similar types of lesions may be repaired with alternative pathways not requiring PCNA

Proliferating cells nuclear antigen complex formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by immunostaining and flow cytometry

The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites was investigated in human quiescent fibroblasts after UV irradiation. Associated PCNA was detected with the monoclonal antibody (MoAb) PC10 and by immunofluorescence assessment with flow cytometry, after a hypotonic lysis step in order to release unbound molecules. Immunofluorescence levels, relatively low in untreated control cells, increased by about threefold after uv irradiation. The time course of PCNA complex formation showed a maximum after about 30 min from irradiation and was found to be dose-dependent up to about 10 J/m2, after that it reached a plateau. Formation of the PCNA-chromatin complex was neither significantly affected by the topoisomerase II inhibitor VP-16, nor by the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide. In contrast, a significant reduction was obtained either after ATP depletion or after incubation with the protein-kinase inhibitor staurosporine. Immunoprecipitation studies on nuclear extracts from 32 P-labeled cells showed that PCNA bound to DNA synthesis sites was phosphorylated. The results indicate that PCNA associated to DNA repair synthesis sites may be detected with PC10 MoAb after a hypotonic lysis step, and provide evidence that the transition from soluble to chromatin-associated form of the protein is dependent on a phosphorylation mechanism

Increased levels of p21CDKN1A do not inhibit the recruitment of NER factors at DNA damage sites.

P21CDK1NA is a cyclin-dependent kinase inhibitor playing multiple roles also in the DNA damage response. Therapeutic trials have been developed to contrast tumor cell proliferation, by exploiting the p21 ability to arrest the cell cycle; in particular, proteasome inhibitors increase p21 protein levels, impairing tumor cell growth. However, this approach is may be potentially dangerous because high p21 levels inhibit the apoptotic response and allow DNA repair, rendering tumor cells resistant to chemotherapy. We have investigated whether the accumulation of p21 levels, induced by the inhibitor of proteasome MG132, may affect nucleotide excision repair (NER) and apoptosis. The results have shown that MG132 induced persistent increased levels of XPC, PCNA and p21 proteins at local DNA damage sites, together with accumulation of XPG, DNA polymerase δ and CAF-1, suggesting that the presence of p21 protein did not block the recruitment of NER factors interacting with PCNA. Immunoprecipitation experiments have shown that DNA pol δ interacts with an ubiquitinated form of p21. These results indicate that p21 regulates steps of NER before degradation

Involvement of the cell cycle inhibitor p21CDKN1A in DNA repair

A variety of chemical and physical agents may induce the formation of different lesions in the DNA molecule. These types of DNA damage may be genotoxic to the cells, and must be removed in order to avoid genomic instability, and to prevent cancer formation. To this end, virtually every organism has developed highly conserved genome surveillance and signaling mechanisms, collectively known as the DNA damage response. This pathway consists of DNA damage signaling cascade (cell cycle checkpoints), and of DNA repair processes able to recognize and remove a great number of DNA lesions. Recent findings have shown that cell cycle checkpoints and DNA repair systems are strictly connected each other. However, the role and the molecular mechanisms underlying these connections are not yet completely understood. Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21CDKN1A plays fundamental roles in the DNA damage response by inducing cell cycle arrest, direct inhibition of DNA synthesis, as well as by regulating transcription and apoptosis. During the last years, several lines of evidence have also indicated that p21 may be directly involved in DNA repair. Participation of p21 in DNA repair pathways, like nucleotide excision repair (NER), and base excision repair (BER), is thought to occur thanks to its interaction with Proliferating Cell Nuclear Antigen (PCNA), a crucial protein involved both in DNA replication and repair. In addition, a direct involvement of p21 in DNA trans-lesion synthesis, has been postulated to keep within low levels the mutagenesis intrinsic in this process. In this review, all relevant findings supporting the participation of p21 protein in NER and BER will be presented. In particular, the ability of p21 to interact with PCNA seems to be required for regulating interaction of DNA repair factors with PCNA. Examples of this role will be discussed together with other aspects of the DNA damage response in which p21 is also involved. A special attention will be given to the dynamics of p21 recruitment to sites of DNA damage. In fact, a common feature of checkpoint and DNA repair factors is their accumulation at nuclear sites where DNA damage has occurred. The involvement of p21 in various DNA repair pathways supports its important function of protein barrier against genome instability

Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts

The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system