The cyclin-dependent kinase inhibitor p21(CDKN1A) as target of anti-cancer drugs

p21(CDKN1A) (WAF1/CIP1/SDI1), the cyclin-dependent kinase (CDK) inhibitor belonging to the Cip/Kip family, was first described as a potent inhibitor of cell proliferation and DNA replication, both in physiological conditions and after DNA damage. More recently, p21 has been recognized to play additional and fundamental roles in other important pathways, including regulation of transcription, apoptosis and DNA repair. Knock-out mouse studies combined with biochemical and functional analysis of cells in culture have indicated a tumor suppressor activity for p21. However, these lines of evidence have been complicated by other findings indicating that p21 can exhibit oncogenic properties. In fact, the evidence that p21 expression may lead to proliferation arrest, is counterbalanced by the rescue of tumor cells from drug-induced apoptosis, and by promoting a metastatic potential. For these reasons, p21 is considered a protein with a dual behavior, with potential benefits, as well as dangerous effects of its expression in malignant cells. Thus, the effectiveness of targeting p21 expression for antitumor therapy needs to be carefully evaluated accordingly. This review summarizes the functions and regulations of p21, and focuses on its involvement in human diseases (particularly cancer), and on the pharmacological approaches to target p21 expression (either positively or negatively) for anticancer therapy. Based on these approaches, the search for new molecules able to promote the tumor-suppressor activity, and/or to interfere with the oncogenic properties of p21, could be promising

Flavonoids uptake and their effect on cell cycle of human colon adenocarcinoma cells (Caco2).

Green tea, mainly through its constituents epigallocatechin gallate, epigallocatechin, epicatechin gallate and epicatechin, has demonstrated anticarcinogenic activity in several animal models, including those for skin, lung and gastro-intestinal tract cancer, although less is known about colorectal cancer. Quercetin, the major flavonoid present in vegetables and fruit, exerts potential anticarcinogenic effects in animal models and cell cultures, but less is known about quercetin glucosides. The objectives of this study were to investigate (i) the antioxidant activity of the phenolic compounds epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside; (ii) the cytotoxicity of different concentrations of epicatechin, epigallocatechin gallate, and gallic acid; (iii) the cellular uptake of epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside and (iv) their effect on the cell cycle. Human colon adenocarcinoma cells were used as experimental model. The results of this study indicate that all dietary flavonoids studied (epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside) show a significant antioxidant effect in a chemical model system, but only epigallocatechin gallate or gallic acid are able to interfere with the cell cycle in Caco2 cell lines. These data suggest that the antioxidant activity of flavonoids is not related to the inhibition of cellular growth. From a structural point of view, the galloyl moiety appears to be required for both the antioxidant and the antiproliferative effects. comCopyright 2002 Cancer Research U

Analysis of p21CDKN1A recruitment to DNA excision repair foci in the UV-induced DNA damage response

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21(waf1/cip1)) is a well known player of the G1 and G2 phase cell cycle checkpoints, which are activated in response to DNA damage. In addition, p21 interacts directly with proliferating cell nuclear antigen (PCNA), thereby inhibiting DNA replication. More controversial is the role of p21 in DNA repair, since both inhibition of and requirement for nucleotide excision repair have been suggested. Since the DNA repair process occurs at discrete nuclear foci in a chromatin-bound compartment, a suitable extraction procedure is necessary to investigate the association of p21 with PCNA in these structures. This chapter focuses on biochemical and immunofluorescence methods to analyze the recruitment of p21 protein to DNA repair foci. Cellular fractionation and subsequent nuclear extraction procedures are described for Western blot analysis of p21 recruitment, as well as for protein-protein interaction studies. An in situ extraction protocol is also described for immunofluorescence microscopy and flow cytometric analyses of nuclear localization and cell cycle distribution of p21 recruited to DNA repair foci. The combination of these methodologies is extremely powerful to investigate in more detail the role of p21 in the UV-induced DNA damage response

Alterazioni della funzione circolatoria: i vasi

La finalità della Patologia generale di trovare una risposta all’antico quesito “come e perché ci si ammala?” pone questa disciplina in una posizione strategica nel percorso formativo di tutte le professioni che si occupano della vita e della salute umana. La Patologia generale si dedica infatti allo studio delle cause e dei meccanismi di malattia, ricercandoli attraverso i vari livelli di complessità dei sistemi vitali, molto spesso intersecando ed integrando conoscenze di altri campi del sapere sicuramente più omogenei. Lo studente troverà molto spesso in questo volume un momento di raccordo ragionato e dinamico tra varie conoscenze acquisite nelle altre discipline già affrontate nel corso del suo progetto formativo. Per coloro che si dedicano allo studio delle scienze della vita, la comprensione dei meccanismi di reazione al danno e degli eventi che si pongono alle basi delle alterazioni delle funzioni generali e specializzate di cellule, tessuti e organi sarà un organico completamento al panorama di conoscenze acquisite; per coloro che, invece, hanno come obbiettivo dedicarsi alla cura o all’assistenza di chi è colpito da quelle alterazioni, questa disciplina “generale” fornirà una solida base cui ancorare la conoscenza “applicabile” delle discipline cliniche, salvaguardando una visione ampia della scienza medica. È a tutti noto, infatti, che in questi ultimi anni le conoscenze nel campo biomedico si sono accresciute a ritmo esponenziale; ciò ha prodotto la frammentazione della scienza medica in numerose specializzazioni con inevitabili conseguenze per cui l’attenzione tende ad essere rivolta più alla malattia che al paziente e l’aggiornamento professionale si presenta sempre più settoriale. Solo una conoscenza della medicina ben radicata su principi e meccanismi generali consentirà al futuro medico di acquisire una forma mentis attenta al paziente e disposta ad accogliere le nuove conoscenze generate dalla ricerca scientifica in un più ampio contesto del sapere medico e biologico

An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites.

Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type

Proliferating cell nuclear antigen bound to DNA synthesis sites: phosphorylation and association with cyclin D1 and cyclin A.

Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G1, or in G2 + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G1/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G1/S phase boundary to drive activation of DNA replication factors

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response

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