Helena L. Borges

Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage

ABSTRACT Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb

The retinoblastoma tumour suppressor protein RB is cleaved by caspases during apoptosis. Here we have mutated the caspase cleavage site in the carboxy terminus of the murine Rb protein in the mouse germ line to create the Rb-MI allele. After endotoxic shock, expression of Rb-MI inhibits apoptosis in the intestines, but not in the spleen, and promotes the survival of male mice. Fibroblasts expressing Rb-MI protein are protected from apoptosis induced by the tumour-necrosis factor-α type I receptor (TNFRI) but remain sensitive to cell death induced by DNA damage. Correspondingly, the release of cytochrome c and the activation of caspase-3 induced by TNFRI, but not by DNA damage, are defective in cells expressing Rb-MI. Our results highlight the importance of Rb cleavage in TNFRI-induced apoptosis.

DNA damage-induced cell death: lessons from the central nervous system

DNA damage can, but does not always, induce cell death. While several pathways linking DNA damage signals to mitochondria-dependent and -independent death machineries have been elucidated, the connectivity of these pathways is subject to regulation by multiple other factors that are not well understood. We have proposed two conceptual models to explain the delayed and variable cell death response to DNA damage: integrative surveillance versus autonomous pathways. In this review, we discuss how these two models may explain the in vivo regulation of cell death induced by ionizing radiation (IR) in the developing central nervous system, where the death response is regulated by radiation dose, cell cycle status and neuronal development.

Radiation-induced apoptosis in developing mouse retina exhibits dose-dependent requirement for ATM phosphorylation of p53

AbstractIonizing radiation (IR) induces DNA breakage to activate cell cycle checkpoints, DNA repair, premature senescence or cell death. A master regulator of cellular responses to IR is the ATM kinase, which phosphorylates a number of downstream effectors, including p53, to inhibit cell cycle progression or to induce apoptosis. ATM phosphorylates p53 directly at Ser15 (Ser18 of mouse p53) and indirectly through other kinases. In this study, we examined the role of ATM and p53 Ser18 phosphorylation in IR-induced retinal apoptosis of neonatal mice. Whole-body irradiation with 2u2009Gy IR induces apoptosis of postmitotic and proliferating cells in the neonatal retinas. This apoptotic response requires ATM, exhibits p53-haploid insufficiency and is defective in mice with the p53S18A allele. At a higher dose of 14u2009Gy, retinal apoptosis still requires ATM and p53 but can proceed without Ser18 phosphorylation. These results suggest that ATM activates the apoptotic function of p53 in vivo through alternative pathways depending on IR dose.

Potentiation of anticancer-drug cytotoxicity by sea anemone pore-forming proteins in human glioblastoma cells

The search for new drugs and treatment approaches is of particular importance for glioblastomas (GBMs), as with other types of malignant gliomas, as they are lethal without the available medical care. Current anticancer cocktails have failed to prolong survival beyond 1 year, in part owing to the natural resistance of GBM cells and to the toxic side effects of the available drugs. In many organisms, cell death can be induced by cytolysins, which are proteins that can form pores in biological membranes. Perhaps by facilitating drugs to enter into the cytosol, cytolysins might be used to increase the efficacy of conventional anticancer agents. Here, the cytotoxicity of two sea anemone pore-forming cytolysins, toxin Bc2, and equinatoxin (EqTx-II) were investigated. Toxin Bc2 and EqTx-II were cytotoxic against human U87 and A172 GBM cell lines either wild type or p53 mutant, a tumor suppressor frequently mutated in malignant gliomas. Moreover, noncytotoxic concentrations of Bc2 or EqTx-II potentiated the cytotoxicity induced by low dose concentrations of all classical chemotherapeutics agents tested: cytosine arabinoside, doxorubicin, and vincristine. In comparison with the cytotoxicity induced by each of these classical anticancer drugs alone, 10–300-fold less of the therapeutic drug was needed when combined with the cytolysins. These results are promising, since lower concentrations of chemotherapeutic drugs could reduce the adverse effects of chemotherapy.

Tumor promotion by caspase-resistant retinoblastoma protein.

The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage.

Reduction of apoptosis in Rb-deficient embryos via Abl knockout.

The retinoblastoma protein RB regulates cell proliferation, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality owing to placental defects that result in excessive apoptosis. RB binds to a number of cellular proteins including the nuclear Abl protein and inhibits its tyrosine kinase activity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. Employing the Rb-null embryos as an in vivo model of apoptosis, we have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system and the embryonic liver. These results are consistent with the inhibitory interaction between RB and Abl, and provide in vivo evidence for the proapoptotic function of Abl.

Simultaneous follow-up of mouse colon lesions by colonoscopy and endoluminal ultrasound biomicroscopy

AIMnTo evaluate the potential use of colonoscopy and endoluminal ultrasonic biomicroscopy (eUBM) to track the progression of mouse colonic lesions.nnnMETHODSnTen mice were treated with a single azoxymethane intraperitoneal injection (week 1) followed by seven days of a dextran sulfate sodium treatment in their drinking water (week 2) to induce inflammation-associated colon tumors. eUBM was performed simultaneously with colonoscopy at weeks 13, 17-20 and 21. A 3.6-F diameter 40 MHz mini-probe catheter was used for eUBM imaging. The ultrasound mini-probe catheter was inserted into the accessory channel of a pediatric flexible bronchofiberscope, allowing simultaneous acquisition of colonoscopic and eUBM images. During image acquisition, the mice were anesthetized with isoflurane and kept in a supine position over a stainless steel heated surgical waterbed at 37 °C. Both eUBM and colonoscopic images were captured and stored when a lesion was detected by colonoscopy or when the eUBM image revealed a modified colon wall anatomy. During the procedure, the colon was irrigated with water that was injected through a flush port on the mini-probe catheter and that acted as the ultrasound coupling medium between the transducer and the colon wall. Once the acquisition of the last eUBM/colonoscopy section for each animal was completed, the colons were fixed, paraffin-embedded, and stained with hematoxylin and eosin. Colon images acquired at the first time-point for each mouse were compared with subsequent eUBM/colonoscopic images of the same sites obtained in the following acquisitions to evaluate lesion progression.nnnRESULTSnAll 10 mice had eUBM and colonoscopic images acquired at week 13 (the first time-point). Two animals died immediately after the first imaging acquisition and, consequently, only 8 mice were subjected to the second eUBM/colonoscopy imaging acquisition (at the second time-point). Due to the advanced stage of colonic tumorigenesis, 5 animals died after the second time-point image acquisition, and thus, only three were subjected to the third eUBM/colonoscopy imaging acquisition (the third time-point). eUBM was able to detect the four layers in healthy segments of colon: the mucosa (the first hyperechoic layer moving away from the mini-probe axis), followed by the muscularis mucosae (hypoechoic), the submucosa (the second hyperechoic layer) and the muscularis externa (the second hypoechoic layer). Hypoechoic regions between the mucosa and the muscularis externa layers represented lymphoid infiltrates, as confirmed by the corresponding histological images. Pedunculated tumors were represented by hyperechoic masses in the mucosa layer. Among the lesions that decreased in size between the first and third time-points, one of the lesions changed from a mucosal hyperplasia with ulceration at the top to a mucosal hyperplasia with lymphoid infiltrate and, finally, to small signs of mucosal hyperplasia and lymphoid infiltrate. In this case, while lesion regression and modification were observable in the eUBM images, colonoscopy was only able to detect the lesion at the first and second time-points, without the capacity to demonstrate the presence of lymphoid infiltrate. Regarding the lesions that increased in size, one of them started as a small elevation in the mucosa layer and progressed to a pedunculated tumor. In this case, while eUBM imaging revealed the lesion at the first time-point, colonoscopy was only able to detect it at the second time-point. All colonic lesions (tumors, lymphoid infiltrate and mucosal thickening) were identified by eUBM, while colonoscopy identified just 76% of them. Colonoscopy identified all of the colonic tumors but failed to diagnose lymphoid infiltrates and increased mucosal thickness and failed to differentiate lymphoid infiltrates from small adenomas. During the observation period, most of the lesions (approximately 67%) increased in size, approximately 14% remained unchanged, and 19% regressed.nnnCONCLUSIONnCombining eUBM with colonoscopy improves the diagnosis and the follow-up of mouse colonic lesions, adding transmural assessment of the bowel wall.

Analysis of tumor morphology and vasculature in an animal model of colorectal cancer using in vivo contrast-enhanced endoluminal ultrasound biomicroscopy

Colorectal cancer (CRC) has a high incidence in the world, being the third leading cause of cancer-related mortality in the United States. Ninety percent of the malignant tumors can be cured if diagnosed in the early stages of localized disease and this motivates a great interest in the development and design of experimental models for CRC detection, staging and monitoring. The aim of this work was to use VEGFR2 (vascular endothelial growth factor receptor 2)-targeted ultrasound contrast agent (UCA) coupled to an endoluminal ultrasound biomicroscopy (eUBM) system to diagnose colon tumors and to analyze tumor vasculature. Colon tumors were induced in mice by using azoxymethane/dextran sulfate sodium and according to histopathology, 55% of colon tumors were adenomas and 45% were adenocarcinomas. Images of the lesion were obtained before and 2-15 minutes after VEGFR2-targeted UCA injection into the mice tail. Compared to post-mortem histopathology, all colonic lesions (lymphoid hyperplasias, ulcerations, thickened mucosa and tumors) were correctly diagnosed by eUBM. In addition, when coupled to a VEGFR2-targeted UCA, the eUBM system provided hyperechoic image areas compatible with intense VEGFR2 staining by immunohistochemical diagnosis. The results suggest that eUBM associated to UCA is a promising tool for histopathologycal and molecular imaging, and could be used to diagnose, stage and monitor colon tumor morphology and vasculature.