Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells.

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase ß and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology.

The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.

Berberine, an epiphany against cancer.

Alkaloids are used in traditional medicine for the treatment of many diseases. These compounds are synthesized in plants as secondary metabolites and have multiple effects on cellular metabolism. Among plant derivatives with biological properties, the isoquinoline quaternary alkaloid berberine possesses a broad range of therapeutic uses against several diseases. In recent years, berberine has been reported to inhibit cell proliferation and to be cytotoxic towards cancer cells. Based on this evidence, many derivatives have been synthesized to improve berberine efficiency and selectivity; the results so far obtained on human cancer cell lines support the idea that they could be promising agents for cancer treatment. The main properties of berberine and derivatives will be illustrated.

Comparison of StemPrintER with Oncotype DX Recurrence Score for predicting risk of breast cancer distant recurrence after endocrine therapy.

OBJECTIVE: Molecular tests predicting the risk of distant recurrence (DR) can be used to assist therapy decision-making in oestrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients after considerations of standard clinical markers. The Oncotype DX Recurrence Score (RS) is a widespread tool used for this purpose. Here, we compared the RS with the StemPrintER Risk Score (SPRS), a novel genomic predictor with a unique biological basis in its ability to measure the expression of cancer stemness genes. MATERIALS AND METHODS: We benchmarked the SPRS vs. RS, alone or in combination with clinicopathological variables expressed by the Clinical Treatment Score (CTS), for the prognostication of DR in a retrospective cohort of 776 postmenopausal patients with ER+/HER2-breast cancer enrolled in the translational arm of the randomised Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. Likelihood ratio (LR) with χ2 test and C-index were used to assess prognostic performance for the entire ten-year follow-up period and in early (0-5 years) and late (5-10 years) intervals. RESULTS: In all patients, the SPRS provided significantly more prognostic information than the RS for ten-year DR prognostication (C-index = 0.688, LR-χ2 = 33.4 vs. C-index = 0.641, LR-χ2 = 22.1) and for late (5-10 years) DR prognostication (C-index = 0.689, LR-χ2 = 18.8 vs. C-index = 0.571, LR-χ2 = 4.7). The SPRS also provided more prognostic information than the RS when added to the CTS in all patients (CTS + SPRS: LR-Δχ2 = 14.9; CTS + RS: LR-Δχ2 = 9.7) and in node-negative patients (CTS + SPRS: LR-Δχ2 = 11.7; CTS + RS: LR-Δχ2 = 6.6). CONCLUSIONS: In postmenopausal ER+/HER2- breast cancer patients, SPRS provided more prognostic information than RS for DR when used alone or in combination with the CTS. The SPRS could therefore potentially identify high-risk patients, who might benefit from aggressive treatments, from low-risk patients who might safely avoid adjuvant chemotherapy or prolongation of endocrine therapy.

Assessing Cell Cycle Independent Function of the CDK Inhibitor p21(CDKN¹A) in DNA Repair.

The cyclin-dependent kinase (CDK) inhibitor p21(CDKN1A) is a small protein that is able to regulate many important cell functions, often independently of its activity of CDK inhibitor. In addition to cell cycle, this protein regulates cell transcription, apoptosis, cell motility, and DNA repair. In particular, p21 may participate in different DNA repair processes, like the nucleotide excision repair (NER), base excision repair (BER), and double-strand breaks (DSB) repair, because of its ability to interact with DNA repair proteins, such as proliferating cell nuclear antigen (PCNA), a master regulator of many DNA transactions. Although this role has been debated for a long time, the influence of p21 in DNA repair has been now established. However, it remain to be clarified how this role is coupled to proteasomal degradation that has been shown to occur after DNA damage. This chapter describes procedures to study p21 protein recruitment to localized DNA damage sites in the cell nucleus. In particular, we describe a technique based on local irrradiation with UV light through a polycarbonate filter with micropores; an in situ lysis procedure to detect chromatin-bound proteins by immunofluorescence; a cell fractionation procedure to study chromatin association of p21 by Western blot analysis, and p21 protein-protein interactions by an immunoprecipitation assay.

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.

The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer.

Accurate detection of altered anaplastic lymphoma kinase (ALK) expression is critical for the selection of lung cancer patients eligible for ALK-targeted therapies. To overcome intrinsic limitations and discrepancies of currently available companion diagnostics for ALK, we developed a simple, affordable and objective PCR-based predictive model for the quantitative measurement of any ALK fusion as well as wild-type ALK upregulation. This method, optimized for low-quantity/-quality RNA from FFPE samples, combines cDNA pre-amplification with ad hoc generated calibration curves. All the models we derived yielded concordant predictions when applied to a cohort of 51 lung tumors, and correctly identified all 17 ALK FISH-positive and 33 of the 34 ALK FISH-negative samples. The one discrepant case was confirmed as positive by IHC, thus raising the accuracy of our test to 100%. Importantly, our method was accurate when using low amounts of input RNA (10 ng), also in FFPE samples with limited tumor cellularity (5-10%) and in FFPE cytology specimens. Thus, our test is an easily implementable diagnostic tool for the rapid, efficacious and cost-effective screening of ALK status in patients with lung cancer.

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase ß, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

Multiple effects of berberine derivatives on colon cancer cells.

The pharmacological use of the plant alkaloid berberine is based on its antibacterial and anti-inflammatory properties; recently, anticancer activity has been attributed to this compound. To exploit this interesting feature, we synthesized three berberine derivatives, namely, NAX012, NAX014, and NAX018, and we tested their effects on two human colon carcinoma cell lines, that is, HCT116 and SW613-B3, which are characterized by wt and mutated p53, respectively. We observed that cell proliferation is more affected by cell treatment with the derivatives than with the lead compound; moreover, the derivatives proved to induce cell cycle arrest and cell death through apoptosis, thus suggesting that they could be promising anticancer drugs. Finally, we detected typical signs of autophagy in cells treated with berberine derivatives.

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.

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG.

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21(CDKN1A), thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3'-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21(-/-) fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA-p21 dependent manner.

Killing of tumor cells: a drama in two acts.

Cancer still represents a major health problem worldwide, which urges the development of more effective strategies. Resistance to chemotherapy, a major obstacle for cancer eradication, is mainly related to an intrinsic failure to activate the apoptotic pathways. However, a protective effect of autophagy toward cancer cells has been recently observed, thus adding further complexity to the development of an effective approach counteracting cancer cell growth and improving the response to therapy.

2-Methoxyestradiol: new perspectives in colon carcinoma treatment.

Colon carcinoma represents a major problem in oncology, since this type of cancer responds poorly to conventional chemotherapy. Many groups are actively involved in the search of new experimental strategies to bypass this problem. We investigated the effects of 2-methoxyestradiol (2-ME), which derives from the NADPH-dependent cytochrome P450 metabolism of 17ß-estradiol. This compound has raised much interest in the past few decades for its inhibitory effects on the growth of cancer cells of different origin; however, little is known about its use on colon carcinoma-derived cell lines. In the present study, we investigated the effects of 2-ME on cell proliferation and cell cycle of two human colon carcinoma cell lines, namely HCT116 and SW613-B3. Our results showed a net anti-proliferative effect of 2-ME on both cell lines, which is accompanied by cell cycle arrest; moreover, we demonstrated that 2-ME is able to induce apoptosis as well as autophagy. This body of evidence points out that 2-ME could be considered as a promising tool against colon carcinoma.

PARP inhibitors: new tools to protect from inflammation.

Poly(ADP-ribosylation) consists in the conversion of ß-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.

p21CDKN1A participates in base excision repair by regulating the activity of poly(ADP-ribose) polymerase-1.

The cell cycle inhibitor p21(CDKN1A) has been shown to participate in nucleotide excision repair by interacting with PCNA. Here we have investigated whether p21 plays a role in base excision repair (BER), by analyzing p21 interactions with BER factors, and by assessing the response of p21(-/-) human fibroblasts to DNA damage induced by alkylating agents. Absence of p21 protein resulted in a higher sensitivity to alkylation-induced DNA damage, as indicated by reduced clonogenic efficiency, defective DNA repair (assessed by the comet test), and by persistence of histone H2AX phosphorylation. To elucidate the mechanisms at the basis of the function of p21 in BER, we focused on its interaction with poly(ADP-ribose) polymerase-1 (PARP-1), an important player in this repair process. p21 was found to bind the automodification/DNA binding domain of PARP-1, although some interaction occurred also with the catalytic domain after DNA damage. This association was necessary to regulate PARP-1 activity since poly(ADP-ribosylation) induced by DNA damage was higher in p21(-/-) human fibroblasts than in parental p21(+/+) cells, and in primary fibroblasts after p21 knock-down by RNA interference. Concomitantly, recruitment of PARP-1 and PCNA to damaged DNA was greater in p21(-/-) than in p21(+/+) fibroblasts. This accumulation resulted in persistent interaction of PARP-1 with BER factors, such as XRCC1 and DNA polymerase beta, suggesting that prolonged association reduced the DNA repair efficiency. These results indicate that p21 regulates the interaction between PARP-1 and BER factors, to promote efficient DNA repair.