Multiple stress signals activate mutant p53 in vivo.

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status.

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity.

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

A high-frequency regulatory polymorphism in the p53 pathway accelerates tumor development.

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele.

A novel A33 promoter-based conditionally replicative adenovirus suppresses tumor growth and eradicates hepatic metastases in human colon cancer models.

PURPOSE: A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC). EXPERIMENTAL DESIGN: We cloned an A33 promoter fragment (A33Pr) that extends from -105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active in CRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo. RESULTS: AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or had minimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs. CONCLUSIONS: These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.

Nitric oxide mediates the suppressive effect of testosterone on cell proliferative response to myelin basic protein.

This study assessed whether the in vitro effect of testosterone on the proliferative response of mononuclear cells to myelin basic protein (MBP) could be mediated by nitric oxide (NO). Testosterone but not cholesterol supplementation specifically suppressed the proliferative response of rat mononuclear cells to MBP and in parallel increased the NO level. NG-monomethyl 1-l-arginine, an inhibitor of NO synthesis, reverted the suppression of the testosterone-induced proliferative response to MBP. These results indicate that changes in the production of NO by testosterone are able to alter the specific T cell proliferation induced by the encephalitogenic MBP and in this way; it could be one of the molecular mechanisms that modulate the development of experimental autoimmune encephalomyelitis (EAE).

Endogenous thyrocyte-produced nitric oxide inhibits iodide uptake and thyroid-specific gene expression in FRTL-5 thyroid cells.

Nitric oxide (NO) is a free radical that mediates a wide array of cell functions. It is generated from l-arginine by NO-synthase (NOS). Expression of NOS isoforms has been demonstrated in thyroid cells. Previous reports indicated that NO donors induce dedifferentiation in thyrocytes. However, the functional significance of endogenous thyrocyte-produced NO has not been explored. This work aimed to study the influence of endogenous NO on parameters of thyroid cell function and differentiation in FRTL-5 cells. We observed that treatment with the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), increased the TSH-stimulated iodide uptake. The TSH-induced sodium iodide symporter (NIS) and thyroglobulin (TG) mRNA expressions were increased after incubation with L-NAME. In transient transfection assays, TSH-stimulated transcriptional activities of NIS and TG promoters were increased by L-NAME. An increment of the TSH-stimulated cell proliferation was observed after NOS inhibition. Similar results were obtained when the action of another NOS inhibitor, N(g)-monomethyl-L-arginine, was analysed for most of these studies. The production of NO, which was not detectable in basal conditions, was increased by TSH. Our data provide strong evidence that endogenous NO could act as a negative signal for TSH-stimulated iodide uptake and thyroid-specific gene expression as well as proliferation in thyrocytes. These findings reveal a possible new inhibitory pathway in the regulation of thyroid cell function.

Oral testosterone in male rats and the development of experimental autoimmune encephalomyelitis.

OBJECTIVES: Considering that sex steroids can influence the immune system, we studied the development of experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune disease of the central nervous system, and the concomitant cell-mediated immunity in gonadally intact and gonadectomized male Wistar rats given testosterone supplementation. METHODS/RESULTS: Sham-operated rats and surgically castrated animals were orally self-administered with vehicle or testosterone added in the water bottle for 20 days before EAE induction. The androgenic effect of oral testosterone self-administration was evidenced by changes in body weight, and in the weights of androgen-dependent testes and seminal vesicles. Testosterone administration reduced the incidence of clinical signs of EAE in sham-operated animals and reversed the clinical symptoms of the disease associated with castrated EAE animals. The clinical signs observed in the different groups correlated with changes in delayed-type hypersensitivity and mononuclear cell-proliferative responses to the encephalitogenic myelin basic protein. Moreover, testosterone but not cholesterol supplementation in vitro suppressed the proliferative response of mononuclear cells to myelin basic protein suggesting that testosterone may affect specific immune functions through direct actions on immune cells. Finally, self-administration of testosterone induced also elevated corticosterone levels that in sham-operated rats correlated with the low incidence of the disease and in gonadectomized animals could be involved in the remission of clinical symptoms of EAE. CONCLUSIONS: These results suggest that orally self-administered testosterone can modulate specific cellular immune responses and serum corticosterone levels leading to changes in the development of EAE.

Age-related changes in the development of experimental autoimmune encephalomyelitis.

A prominent feature of multiple sclerosis is its high incidence of onset in the third decade of life and its relatively rare onset in persons older than 50 years. In order to study age-related restriction of clinical expression, a comparative biochemical, immunological and histological study was undertaken during development of experimental autoimmune encephalomyelitis (EAE) in young (7 weeks) and middle-aged (15 months) Wistar rats. Young rats showed characteristic clinical signs 12-16 days postinduction, and then they spontaneously recuperated. In middle-aged rats, the incidence of clinical signs was significantly reduced, with a later onset of the disease. Similar biochemical and histological alterations were detected in both age groups, but they were present in a later stage in middle-aged animals. However, cellular and humoral immune responses to myelin basic protein (MBP) were observed 15 days postinduction in all EAE animals. The study of anti-MBP IgG isotype pattern in 7-week-old animals indicated a predominant Th1-type immune response during the acute stage of EAE, with antibodies predominantly recognizing the MBP 96-128 peptide. In contrast, 15-month-old animals showed a less prominent Th1 response, without any epitope dominance. The changes in immune function found in middle-aged animals may account for the different susceptibility and expression of EAE, and may also be relevant to the different clinical expression observed in multiple sclerosis with maturation.

Serum testosterone and corticosterone levels in acute experimental autoimmune encephalomyelitis (EAE) in male Wistar rats.

OBJECTIVES: Experimental autoimmune encephalomyelitis (EAE) was induced to investigate the levels of circulating total testosterone (TT) and the possible association of corticosterone with the steroid-producing capacity of the testes. SETTING AND DESIGN: We determined gonad weights, serum TT and corticosterone levels during the development of EAE in male rats. METHODS: Active EAE was induced in young male Wistar rats by injection of whole myelin in complete Freund's adjuvant. All the rats were weighted and monitored daily for clinical signs and blooded during different phases of the disease. Serum TT was measured by radioimmunoassay and circulating corticosterone was determined using a competitive enzyme immunoassay. Also testes and seminal vesicles were removed to determine their weights. RESULTS: Seminal vesicle weights and serum TT levels diminished during the acute stage of the disease in all EAE rats and then they began gradually to increase, reaching the normal values at the post-recovery phase. Concomitantly a significant increase in serum corticosterone levels was observed during the acute EAE and the post-recovery phase was accompanied by a decline in corticosterone levels. MAIN FINDING: Our results indicated an inverse correlation between serum TT and corticosterone levels during the acute EAE. Moreover, the diminution of TT was not a consequence of an alteration of the testes induced by anti-myelin antibodies neither contributed by apoptosis of testis cells by exposure to corticosterone. CONCLUSIONS: The negative correlation between corticosterone and TT levels associated to EAE may be relevant in understanding the association of the endocrine system and the development of autoimmune demyelinating diseases.

Interaction between gonadal steroids and neuroimmune system in acute experimental autoimmune encephalomyelitis (EAE) in Wistar rats.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T lymphocytes directed against myelin antigens. Since neuroendocrine-immune dysfunction appears to contribute to the pathogenesis of autoimmune diseases, the present work was designed to study the effect of changes in the endocrine system on the development of acute EAE and the immune response against myelin basic protein (MBP). Intact and sham males and intact female Wistar rats showed the most severe clinical symptoms (acute period) 12-14 days post-inoculation (dpi). Then, they began gradually to recover, regaining the total ability to walk by 15-17 dpi. Male Wistar rats with altered levels of gonadal hormones by surgical castration showed an onset of the symptoms retarded 2-3 days with respect to the other EAE groups, showing neuropathological symptoms up to 27-28 dpi, and remaining with lower body weight even at 40 dpi. The castrated animals exhibited a specific delay in MBP-stimulated DTH reactivity that correlates with the delay in the onset of the clinical symptoms. Also significant lymphocyte proliferation to MBP was still present at 35 dpi that was absent in the sham group. The distribution of the IgG subclasses indicated that at 35 dpi castrated animals have a higher IgG2b/IgG1 ratio (35.1) in comparison to that presented by sham rats (4.8). Considering that at this time the castrated animals were not completely recuperated, these results could indicate an ongoing inflammatory immune response associated with Th1 activity in these animals. Also castrated animals developed antibodies to a diversity of MBP epitopes in comparison to sham rats, which presented a dominance of antibodies to MBP peptide p96-128. These results indicate that sex hormones levels regulate cell-mediated immunity and the specificity of anti-MBP antibodies related to the induction and development of acute EAE.