A.Ivana Scovassi

Multiple roles of the cell cycle inhibitor p21CDKN1A 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.

Berberine: New perspectives for old remedies

Chemical compounds derived from plants have been used since the origin of human beings to counteract a number of diseases. Among them, the natural isoquinoline alkaloid berberine has been employed in Ayurvedic and Chinese Medicine for hundreds of years with a wide range of pharmacological and biochemical effects. More recently, a growing body of reports supports the evidence that berberine has anticancer effects, being able to block the proliferation of and to kill cancer cells. This review addresses the properties and therapeutic use of berberine and focuses on the recent advances as promising anticancer drug lead.

Conversation between apoptosis and autophagy: "Is it your turn or mine?".

Drug resistance of cancer cells is often correlated with apoptosis evasion; however, an active involvement of autophagy in this scenario has been recently proposed, based on the evidence that autophagy could exert a protective role toward the activation of apoptosis in cancer cells. In this review, we briefly review the basic features of apoptosis, and we describe in details the molecular patterns of autophagy, with a special emphasis on its still controversial physiological function(s). The crucial factors governing the cross talk between autophagy and apoptosis will be illustrated.

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase δ assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N′-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

PARP inhibitors: New tools to protect from inflammation

Poly(ADP-ribosylation) consists in the conversion of β-NAD(+) into ADP-ribose, which is then bound to acceptor proteins and further used to form polymers of variable length and structure. The correct turnover of poly(ADP-ribose) is ensured by the concerted action of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) enzymes, which are responsible for polymer synthesis and degradation, respectively. Despite the positive role of poly(ADP-ribosylation) in sensing and repairing DNA damage, generated also by ROS, PARP over-activation could allow NAD depletion and consequent necrosis, thus leading to an inflammatory condition in many diseases. In this respect, inhibition of PARP enzymes could exert a protective role towards a number of pathological conditions; i.e. the combined treatment of tumors with PARP inhibitors/anticancer agents proved to have a beneficial effect in cancer therapy. Thus, pharmacological inactivation of poly(ADP-ribosylation) could represent a novel therapeutic strategy to limit cellular injury and to attenuate the inflammatory processes that characterize many disorders.

Environmental immune disruptors, inflammation and cancer risk

An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c- myc gene

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosis-prone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.

Causes of genome instability: the effect of low dose chemical exposures in modern society

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genomes integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Poly(ADPR) polymerase-1 and poly(ADPR) glycohydrolase level and distribution in differentiating rat germinal cells

Poly(ADP-ribose)polymerase (PARP-1) and poly(ADP-ribose)glycohydrolase (PARG) are responsible for the transient poly(ADP-ribosyl)ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly(ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

The cell-cycle inhibitor p21CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.