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Recent epidemiological evidence suggests that exposure to antibiotics in early-to-middle adulthood is associated with an increased risk of colorectal adenoma. However, mechanistic studies in established preclinical cancer to examine these claims are extremely limited. Therefore, we investigated the effect of long-term exposure of an antibiotic cocktail composed of Vancomycin, Neomycin, and Streptomycin, on tumor development and progression in the Apc Min/+ mouse, an established genetic model for familial adenomatous polyposis. Clinical pathologies related to tumor development as well as intestinal and colon tissue histopathology were studied at ages 8, 12, and 16 weeks of age, which correspond to the approximate ages of development of neoplasia, gut inflammation with polyposis, and cancer progression, respectively, in this animal model. We show that the antibiotics significantly increase the severity of clinical symptoms, including effects on intestinal histology and goblet cell numbers. In addition, they promote small intestinal polyposis. Finally, metagenomic analysis of fecal samples demonstrated that antibiotic exposure is associated with a significant but nonuniform depletion of the animal's natural gut flora. Overall, these findings support the premise that long-term antibiotic exposure mediates the selected depletion of gut microbial communities and the concomitant thinning of the protective mucus layer, resulting in an increase in tumor development., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The cGMP signaling axis has been implicated in the suppression of intestinal cancers, but the inhibitory mechanism and the extent to which this pathway can be targeted remains poorly understood. This study has tested the effect of cGMP-elevating agents on tumorigenesis in the Apc Min /+ mouse model of intestinal cancer. Treatment of Apc Min/+ mice with the receptor guanylyl-cyclase C (GCC) agonist linaclotide, or the phosphodiesterase-5 (PDE5) inhibitor sildenafil, significantly reduced the number of polyps per mouse (67% and 50%, respectively). Neither of the drugs affected mean polyp size, or the rates of apoptosis and proliferation. This was possibly due to increased PDE10 expression, as endogenous GCC ligands were not deficient in established polyps. These results indicated that the ability of these drugs to reduce polyp multiplicity was primarily due to an effect on nonneoplastic tissues. In support of this idea, Apc Min /+ mice exhibited reduced levels of endogenous GCC agonists in the nonneoplastic intestinal mucosa compared with wild-type animals, and this was associated with crypt hyperplasia and a loss of goblet cells. Administration of either sildenafil or linaclotide suppressed proliferation, and increased both goblet cell numbers and luminal apoptosis in the intestinal mucosa. Taken together, the results demonstrate that targeting cGMP with either PDE5 inhibitors or GCC agonists alters epithelial homeostasis in a manner that reduces neoplasia, and suggests that this could be a viable chemoprevention strategy for patients at high risk of developing colorectal cancer. Cancer Prev Res; 11(2); 81-92. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Recent clinical studies found a strong association of colonic inflammation and Inflammatory bowel disease (IBD)-like phenotype with NonAlcoholic Fatty liver Disease (NAFLD) yet the mechanisms remain unknown. The present study identifies high mobility group box 1 (HMGB1) as a key mediator of intestinal inflammation in NAFLD and outlines a detailed redox signaling mechanism for such a pathway. NAFLD mice showed liver damage and release of elevated HMGB1 in systemic circulation and increased intestinal tyrosine nitration that was dependent on NADPH oxidase. Intestines from NAFLD mice showed higher Toll like receptor 4 (TLR4) activation and proinflammatory cytokine release, an outcome strongly dependent on the existence of NAFLD pathology and NADPH oxidase. Mechanistically intestinal epithelial cells showed the HMGB1 activation of TLR-4 was both NADPH oxidase and peroxynitrite dependent with the latter being formed by the activation of NADPH oxidase. Proinflammatory cytokine production was significantly blocked by the specific peroxynitrite scavenger phenyl boronic acid (FBA), AKT inhibition and NADPH oxidase inhibitor Apocynin suggesting NADPH oxidase-dependent peroxynitrite is a key mediator in TLR-4 activation and cytokine release via an AKT dependent pathway. Studies to ascertain the mechanism of HMGB1-mediated NADPH oxidase activation showed a distinct role of Receptor for advanced glycation end products (RAGE) as the use of inhibitors targeted against RAGE or use of deformed HMGB1 protein prevented NADPH oxidase activation, peroxynitrite formation, TLR4 activation and finally cytokine release. Thus, in conclusion the present study identifies a novel role of HMGB1 mediated inflammatory pathway that is RAGE and redox signaling dependent and helps promote ectopic intestinal inflammation in NAFLD., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis and has been implicated in the suppression of colitis and colon cancer. In this study, we investigated the cGMP-elevating ability of the phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent disease in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against dextran-sulfate sodium (DSS)-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared with untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared with polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration and reduced expression of iNOS, IFNγ, and IL6 compared with untreated controls. Most of the protection conferred by sildenafil was during the initiation stage of carcinogenesis (38% reduction in multiplicity). Administration of sildenafil during the later promotion stages did not affect multiplicity but had a similar effect on the polyp phenotype, including increased mucus production, and reduced proliferation and inflammation. In summary, the results demonstrate that oral administration of sildenafil suppresses polyp formation and inflammation in mice treated with AOM/DSS. This validation of PDE5 as a target highlights the potential therapeutic value of PDE5 inhibitors for the prevention of colitis-driven colon cancer in humans. Cancer Prev Res; 10(7); 377-88. ©2017 AACR See related editorial by Piazza, p. 373 ., (©2017 American Association for Cancer Research.)

Objective: To determine whether, given a limited budget, a state's low-income uninsured population would have greater benefit from a colorectal cancer (CRC) screening program using colonoscopy or fecal immunochemical testing (FIT)., Data Sources/study Setting: South Carolina's low-income, uninsured population., Study Design: Comparative effectiveness analysis using microsimulation modeling to estimate the number of individuals screened, CRC cases prevented, CRC deaths prevented, and life-years gained from a screening program using colonoscopy versus a program using annual FIT in South Carolina's low-income, uninsured population. This analysis assumed an annual budget of $1 million and a budget availability of 2 years as a base case., Principal Findings: The annual FIT screening program resulted in nearly eight times more individuals being screened, and more important, approximately four times as many CRC deaths prevented and life-years gained than the colonoscopy screening program. Our results were robust for assumptions concerning economic perspective and the target population, and they may therefore be generalized to other states and populations., Conclusions: A FIT screening program will prevent more CRC deaths than a colonoscopy-based program when a state's budget for CRC screening supports screening of only a fraction of the target population., (© Health Research and Educational Trust.)

Purpose: African-Americans (AA) have a higher incidence of and lower survival from colorectal cancer (CRC) compared with European Americans (EA). In the present study, statewide, population-based data from South Carolina Central Cancer Registry are used to investigate the relationship between race and age on advanced-stage CRC survival., Methods: The study population was comprised of 3,865 advanced pathologically documented colon and rectal adenocarcinoma cases diagnosed between 01 January 1996 and 31 December 2006: 2,673 (69 %) EA and 1,192 (31 %) AA. Kaplan-Meier methods were used to generate median survival time and corresponding 95 % confidence intervals (CI) by race, age, and gender. Factors associated with survival were evaluated by fitting Cox proportional hazards regression models to generate hazard ratios (HR) and 95 % CI., Results: We observed a significant interaction between race and age on CRC survival (p = 0.04). Among younger patients (<50 years), AA race was associated with a 1.34 times (95 % CI 1.06-1.71) higher risk of death compared with EA. Among older patients, we observed a modest increase in risk of death among AA men compared with EA [HR 1.16 (95 % CI 1.01-1.32)] but no difference by race between women [HR 0.94 (95 % CI 0.82-1.08)]. Moreover, we observed that the disparity in survival has worsened over the past 15 years., Conclusions: Future studies that integrate clinical, molecular, and treatment-related data are needed for advancing understanding of the racial disparity in CRC survival, especially for those <50 years old.

TS (thymidylate synthase) is a key enzyme in the de novo biosynthesis of dTMP, and is indispensable for DNA replication. Previous studies have shown that intracellular degradation of the human enzyme [hTS (human thymidylate synthase)] is mediated by the 26S proteasome, and occurs in a ubiquitin-independent manner. Degradation of hTS is governed by a degron that is located at the polypeptide's N-terminus that is capable of promoting the destabilization of heterologous proteins to which it is attached. The hTS degron is bipartite, consisting of two subdomains: an IDR (intrinsically disordered region) that is highly divergent among mammalian species, followed by a conserved amphipathic α-helix (designated hA). In the present report, we have characterized the structure and function of the hTS degron in more detail. We have conducted a bioinformatic analysis of interspecies sequence variation exhibited by the IDR, and find that its hypervariability is not due to diversifying (or positive) selection; rather, it has been subjected to purifying (or negative) selection, although the intensity of such selection is relaxed or weakened compared with that exerted on the rest of the molecule. In addition, we have verified that both subdomains of the hTS degron are required for full activity. Furthermore, their co-operation does not necessitate that they are juxtaposed, but is maintained when they are physically separated. Finally, we have identified a 'cryptic' degron at the C-terminus of hTS, which is activated by the N-terminal degron and appears to function only under certain circumstances; its role in TS metabolism is not known.

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, β-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or β-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-β, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1β and IL-6 in intestinal tissue and IL-1β and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49f(high)CD61(high)-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-β (TGFβ) signaling and exhibited gene expression signatures of stemness, TGFβ signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs.

The 26 S proteasomal complex, which is responsible for the bulk of protein degradation within the cell, recognizes its target substrates via covalently linked polyubiquitin moieties. However, a small but growing number of proteasomal substrates are degraded without a requirement for ubiquitinylation. One such substrate is the pyrimidine biosynthetic enzyme thymidylate synthase (EC 2.1.1.45), which catalyzes the synthesis of TMP and is the sole de novo source of TTP for DNA replication and repair. Previous work showed that intracellular proteolysis of human thymidylate synthase is directed by a degron at the polypeptide's N-terminal end, composed of an intrinsically disordered region (IDR) followed by a highly conserved amphipathic α-helix (hA). In the present report, we show that the hA helix does not function simply as an extension or scaffold for the IDR; rather, it provides a specific structural component that is necessary for degradation. Furthermore, its helical conformation is required for this function. We demonstrate that small domains from heterologous proteins can substitute for the IDR and the hA helix of human thymidylate synthase, indicating that the degradation-promoting function of these regions is not sequence-specific. The results, in general, indicate that cooperation between intrinsically disordered domains and α-helical segments is required for ubiquitin-independent degradation by the proteasome. There appears to be little sequence constraint on the ability of these regions to function as degron constituents. Rather, it is the overall conformation (or lack thereof) that is critical.

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Criteria for diagnosing cachexia in adults include unintentional loss in body weight, decreased strength, fatigue, anorexia, and low muscle mass. Cachexia is also associated with systemic inflammation, altered metabolism, and anemia. The Apc(Min/+) mouse is a model of cachexia directly related to intestinal tumor burden and subsequent chronic inflammation. These mice also demonstrate muscle weakness, fatigue, decreased volitional activity, and elevated circulating IL-6 levels. The purpose of this study was to determine the time course of changes in physical activity and their relationship to anemia, muscle apoptosis, and muscle mass and body mass loss during cachexia. A subset of male Apc(Min/+) mice were given access to voluntary activity wheels from 5 to 26 wk of age, while sedentary male Apc(Min/+) mice were housed in cages lacking wheels. At the study's end mice were stratified by cachectic symptoms. Severely cachectic mice had decreased wheel running performance at 15 wk of age, while anemia and body weight loss were not present until 18 wk of age. Severely cachectic mice had lower hemoglobin levels compared with mildly cachectic mice at 13, 18, and 22 wk of age. Severely cachectic mice also demonstrated threefold more BCL2-associated X protein (BAX) protein in the gastrocnemius muscle at 26 wk of age compared with mildly cachectic mice. In sedentary Apc(Min/+) mice at 26 wk of age anemia was present, and markers of apoptosis were induced in severely cachectic muscle. Proapoptotic protein expression was induced in both red and white portions of gastrocnemius muscle as well as in soleus muscle of severely cachectic mice compared with mildly cachectic mice. These data demonstrate that decrements in wheel running performance precede loss of body mass and that inherent muscle oxidative capacity is not protective against muscle apoptosis.

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis., (©2010 AACR.)

The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an alpha-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N(alpha)-acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.

Hemoglobin (Hb) uniquely associates with proinflammatory HDL in atherogenic mice and coronary heart disease (CHD) patients. In this paper, we report that Hb and its scavenger proteins, haptoglobin (Hp) and hemopexin (Hx) are significantly increased in apoA-1-containing particles of HDL both in mouse models of hyperlipidemia and in CHD patients, when compared with wild type mice and healthy donors, respectively. We further demonstrate that the association of Hb, Hp, and Hx proteins with HDL positively correlates with inflammatory properties of HDL and systemic inflammation in CHD patients. Interestingly, HDL from Hp(-/-) mice under atherogenic conditions does not accumulate Hb and is anti-inflammatory, suggesting that (i) Hp is required for the association of Hb with HDL and (ii) Hb x Hp complexes regulate the inflammatory properties of HDL. Moreover, treatment of apoE(-/-) mice with an apoA-1 mimetic peptide resulted in significant dissociation of Hb x Hp complexes from HDL and improvement of HDL inflammatory properties. Our data strongly suggest that HDL can become proinflammatory via the Hb x Hp pathway in mice and humans, and dissociation of Hb x Hp x Hx complexes from apoA-1-containing particles of HDL may be a novel target for the treatment of CHD.

Diet and exercise are two environmental factors that can alter colon cancer risk. The purpose of this study was to determine if regular moderate-intensity treadmill exercise training could attenuate polyp formation in Apc(Min/+) mice fed the Western-style diet. Four-week-old male Apc(Min/+) mice (n = 12 per group) were assigned to AIN-76A Control, AIN-76A Exercise, Western Control, or Western Exercise treatment groups. Mice were weaned to these diets and either subjected to regular moderate-intensity treadmill exercise (18 m/min, 60 min/d, 6 d/wk) or remained sedentary for 6 weeks. Mice fed the Western-style diet consumed approximately 14% more calories and had 42% more epididymal fat compared with mice fed the AIN-76A diet. Exercise had no effect on fat pad mass with either diet treatment. Exercise reduced total intestinal polyp number by 50% and the number of large polyps (>1 mm diameter) by 67% in AIN-76A-fed mice. The Western-style diet increased polyp number by 75% when compared with AIN-76A-fed mice, but exercise did not decrease polyp number or alter polyp size in mice fed the Western-style diet. Markers of systemic inflammation and immune system function were improved with exercise in mice fed the AIN-76A diet. Mice fed the Western-style diet showed more inflammation and immunosuppression, which were not completely ameliorated by exercise. These data suggest that the induction of adiposity, inflammation, and immunosuppression by the Western-style diet may compromise the beneficial effect of moderate-intensity exercise on the intestinal polyp burden in Apc(Min/+) mice.

Colorectal cancer risk is increased in shift workers with presumed circadian disruption. Intestinal epithelial cell proliferation is gated throughout each day by the circadian clock. Period 2 (Per2) is a key circadian clock gene. Per2 mutant (Per2(m/m)) mice show an increase in lymphomas and deregulated expression of cyclin D and c-Myc genes that are key to proliferation control. We asked whether Per2 clock gene inactivation would accelerate intestinal and colonic tumorigenesis. The effects of PER2 on cell proliferation and beta-catenin were studied in colon cancer cell lines by its down-regulation following RNA interference. The effects of Per2 inactivation in vivo on beta-catenin and on intestinal and colonic polyp formation were studied in mice with Per2 mutation alone and in combination with an Apc mutation using polyp-prone Apc(Min/+) mice. Down-regulation of PER2 in colon cell lines (HCT116 and SW480) increases beta-catenin, cyclin D, and cell proliferation. Down-regulation of beta-catenin along with Per2 blocks the increase in cyclin D and cell proliferation. Per2(m/m) mice develop colonic polyps and show an increase in small intestinal mucosa beta-catenin and cyclin D protein levels compared with wild-type mice. Apc(Min/+)Per2(m/m) mice develop twice the number of small intestinal and colonic polyps, with more severe anemia and splenomegaly, compared with Apc(Min/+) mice. These data suggest that Per2 gene product suppresses tumorigenesis in the small intestine and colon by down-regulation of beta-catenin and beta-catenin target genes, and this circadian core clock gene may represent a novel target for colorectal cancer prevention and control.

The contribution of acute phase plasma proteins to host immune responses remains poorly characterized. To better understand the role of the acute phase reactant and major hemoglobin-binding protein haptoglobin (Hp) on the function of immune cells, we generated Hp-deficient C57BL/6J mice. These mice exhibit stunted development of lymphoid organs associated with lower counts of mature T and B cells in the blood and secondary lymphoid compartments. Moreover, these mice show markedly reduced adaptive immune responses as represented by reduced accumulation of IgG antibody after immunization with adjuvant and nominal antigen, abrogation of Th1-dominated delayed-type hypersensitivity reaction, loss of mitogenic responses mounted by T cells, and reduced T cell responses conveyed by APCs. Collectively, these defects are in agreement with the observations that Hp-deficient mice are not capable of generating a recall response or deterring a Salmonella infection as well as failing to generate tumor antigen-specific responses. The administration of Hp to lymphocytes in tissue culture partially ameliorates these functional defects, lending further support to our contention that the acute phase response protein Hp has the ability to regulate immune cell responses and host immunity. The phenotype of Hp-deficient mice suggests a major regulatory activity for Hp in supporting proliferation and functional differentiation of B and T cells as part of homeostasis and in response to antigen stimulation.

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.

Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.

Objectives: We sought to understand the importance of oxidative stress in explaining why the haptoglobin (Hp) genotype determines myocardial infarction (MI) size in diabetes mellitus (DM)., Background: Two common alleles (1 and 2) exist at the Hp locus in humans. The Hp 2 allele is associated with increased MI size in individuals with DM. In vitro, the Hp 2 protein is associated with increased generation of oxidatively active iron, whereas the Hp 1 protein is associated with increased production of the antioxidant cytokine interleukin (IL)-10., Methods: Myocardial infarction was produced by myocardial ischemia-reperfusion (IR) in DM C57BL/6 mice carrying the Hp 1 or Hp 2 allele. Myocardial oxidative stress after IR was assessed using electrospray ionization mass spectrometry. Redox active iron and IL-10 were measured in the serum after IR., Results: Myocardial infarction size was significantly larger in Hp 2 mice as compared with Hp 1 mice (44.3 +/- 9.3% vs. 21.0 +/- 4.0%, p = 0.03), and these larger infarctions were associated with a significant increase in a panel of hydroxyl-eicosatetraenoic acids. Redox active iron was greater in Hp 2 mice (0.45 +/- 0.11 micromol/l vs. 0.14 +/- 0.05 micromol/l, p = 0.02), whereas IL-10 was greater in Hp 1 mice (85.8 +/- 12.9 pg/microl vs. 46.7 +/- 10.8 pg/microl, p = 0.04) after IR. Administration of an antioxidant (BXT-51072) to Hp 2 mice reduced myocardial injury after IR by more than 80% (p = 0.003), but no myocardial protection was provided by the antioxidant to Hp 1 mice., Conclusions: The increased MI size in DM Hp 2 mice occurring after IR may be due to increased oxidative stress.

Germ-line mutation of the Apc gene has been linked to familial adenomatous polyposis (FAP) that predisposes to colon cancer. Apc(Min/+) mice, heterozygous for the Apc gene mutation, progressively develop small intestinal tumours in a manner that is analogous to that observed in the colon of patients with FAP (Su et al. 1992; Fodde et al. 1994; Moser et al. 1995). We have studied the effects of Apc gene mutation on murine intestinal and extra-intestinal, proliferatively active tissues. We have contrasted the histology to that of the age- and sex-matched wild-type C57BL/6 mice. Histological assessment of the normal appearing intestinal mucosa demonstrates minimal change in size of crypts. In contrast, villi are longer in the ileum of Apc(Min/+) mice relative to C57BL/6 mice at 12 and 15 weeks of age. Vigorous splenic haematopoiesis in Apc(Min/+) mice was seen at 12 and 15 weeks of age, as reflected by marked splenomegaly, increased splenic haematopoietic cells and megakaryocytes. Peripheral blood counts, however, did not differ between C57BL/6 and Apc(Min/+) mice at 15 weeks of age. Lymphoid depletion in Apc(Min/+) mice was characterized by diminished numbers of splenic lymphoid follicles and small intestinal Peyer's patches. The ovaries of 12- and 15-week-old Apc(Min/+) mice exhibited increased numbers of atretic follicles, and estrous cycling by serial vaginal smears showed tendency of elongation in the mutant mice during these age ranges. The testicles of 10-week-old Apc(Min/+) mice showed increased numbers of underdeveloped seminiferous tubules. Collectively, these data suggest that, in addition to its obvious effects upon intestinal adenoma formation, Apc gene mutation causes impairment of developmental and apparent differentiation blockade in proliferative tissues, including those of the haematopoietic system, lymphoid and reproductive tract.

Thymidylate synthase (TS) catalyses the reductive methylation of dUMP to form dTMP, a reaction that is essential for maintenance of nucleotide pools during cell growth. Because the enzyme is indispensable for DNA replication in actively dividing cells, it is an important target for cytotoxic drugs used in cancer chemotherapy, including fluoropyrimidines (e.g. 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and anti-folates (e.g. raltitrexed, LY231514, ZD9331 and BW1843U89). These drugs generate metabolites that bind to the enzyme's active site and inhibit catalytic activity, leading to thymidylate deprivation and cellular apoptosis. Ligand binding to TS results in stabilization of the enzyme and an increase in its intracellular concentration. Previously, we showed that degradation of the TS polypeptide is carried out by the 26 S proteasome in a ubiquitin-independent manner. Such degradation is directed by the disordered N-terminal region of the TS polypeptide, and is abrogated by ligand binding. In the present study, we have verified the ubiquitin-independent nature of TS proteolysis by showing that a 'lysine-less' polypeptide, in which all lysine residues were replaced by arginine, is still subject to proteasome-mediated degradation. In addition, we have mapped the structural determinants of intracellular TS degradation in more detail and show that residues at the N-terminal end of the molecule, particularly the penultimate amino acid Pro2, play an important role in governing the half-life of the enzyme. This region is capable on its own of destabilizing an evolutionarily distinct TS molecule that normally lacks this domain, indicating that it functions as a degradation signal. Interestingly, degradation of an intrinsically unstable mutant form of TS, containing a Pro-->Leu substitution at residue 303, is directed by C-terminal, rather than N-terminal, sequences. The implications of these findings for the control of TS expression, and for the regulation of protein degradation in general, are discussed.

Cachexia is characterized as an inflammatory state induced by the cancer environment, which is accompanied by the loss of muscle and fat mass. Well-investigated mechanisms of cachexia include the suppression of myofiber protein synthesis and the induction of the protein degradation. However, it is not well characterized whether chronic inflammation during cachexia induces myofiber degeneration, which contributes to muscle mass loss and decreased functional capacity. The purpose of this study was to determine whether Apc(Min/+) mice, which demonstrate a chronic systemic inflammatory state due to an intestinal tumor burden, undergo cachexia and whether the myofibers exhibit signs of degeneration and/or regeneration. Six-month-old female Apc(Min/+) body weight decreased 21% compared with C57BL/6 mice and was not the result of blunted growth. Apc(Min/+) gastrocnemius muscle was reduced 45%, and soleus mean fiber cross-sectional area decreased 24% vs. C57BL/6 mice. Soleus muscle morphology demonstrated pathology of myofibers undergoing degeneration and/or regeneration. These data demonstrate that the Apc(Min/+) mouse becomes cachectic by 6 mo of age and that skeletal muscle degeneration and regeneration may be related to the muscle loss.

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.

Moderate-intensity treadmill running can alter male Apc(Min/+) mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc(Min/+) mouse intestinal polyp development in male and female mice. Male and female Apc(Min/+) mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 +/- 9; P = 0.038) compared with male controls (93 +/- 7). The number of large polyps (>/=1-mm diameter) were also reduced by 38% in male treadmill runners (49 +/- 6; P = 0.005) compared with male controls (79 +/- 6). Treadmill running in female Apc(Min/+) mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 +/- 9 mg; P = 0.011) and wheel runners (75 +/- 6 mg; P = 0.004) compared with controls (141 +/- 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 +/- 0.6 pg/ml) and 78% in male wheel runners (6.6 +/- 3.3 pg/ml) compared with control mice (27.9 +/- 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 +/- 1.2 pg/ml) and 90% decrease with wheel running (2.9 +/- 2.0 pg/ml) compared with control mice (21.1 +/- 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 (P = 0.024) and 24% (P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.

We report that haptoglobin, an acute-phase protein produced by liver cells in response to interleukin-6 (IL-6), can modulate the inflammatory response induced by endotoxins. We provide evidence that haptoglobin has the ability to selectively antagonize lipopolysaccharide (LPS) effects in vitro by suppressing monocyte production of tumour necrosis factor-alpha, IL-10 and IL-12, while it fails to inhibit the production of IL-6, IL-8 and IL-1 receptor antagonist. In two animal models of LPS-induced bronchopulmonary hyperreactivity and endotoxic shock, haptoglobin knockout mice were more sensitive to LPS effects compared to their wild-type counterparts. The present data suggest that haptoglobin regulates monocyte activation following LPS stimulation. The increase in haptoglobin levels during an acute-phase reaction may generate a feedback effect which dampens the severity of cytokine release and protects against endotoxin-induced effects.

The biological functions of the acute- phase protein haptoglobin (Hp) may be related to its ability to bind hemoglobin (Hb) or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. With the use of transgenic mice overexpressing Hp in alveolar macrophages, we demonstrated that Hp diminished Hb-induced lung injury when the lung was exposed to whole blood. In transgenic mouse lungs, Hb was more efficiently removed, and the induction of stress- responsive heme oxygenase-1 gene was significantly lower when compared with wild-type mice. At 24 h after blood treatment, the ferritin level that serves as an index for intracellular iron content was also lower in alveolar macrophages in transgenic mice than in wild-type mice. We propose that an Hp-mediated Hb catabolism process exists in alveolar macrophages. This process is likely coupled to an iron mobilization pathway and may be an efficient mechanism to reduce oxidative damage associated with hemolysis.

Intravascular hemolysis is associated with several pathologic conditions that include hemoglobinopathies, trauma, malaria, and bacterial infections. Among plasma-protective proteins against oxidative damage caused by red blood cell rupture, haptoglobin and hemopexin are thought to play a crucial role. Haptoglobin and hemopexin, by binding with high-affinity hemoglobin and heme, respectively, exert an antioxidant action by preventing heme-catalyzed free radical production. Moreover, these proteins prevent iron loss by inhibiting glomerular filtration of hemoglobin and heme diffusion through plasma membranes. Analysis of single-null mice demonstrated the antioxidant action of haptoglobin and hemopexin in vivo and suggests that the 2 proteins cooperate in the resolution of hemolytic stress. To evaluate the physiological relevance of the haptoglobin-hemopexin system and the principal targets of its action, we generated haptoglobin-hemopexin double-knockout mice and analyzed them under basal conditions and after acute hemolysis. Whereas haptoglobin-hemopexin double-null mice displayed no obvious alteration in phenotype under basal conditions, nonlethal hemolytic stress in these animals led to pronounced splenomegaly as well as liver inflammation and fibrosis. These data demonstrate that haptoglobin and hemopexin together are essential for protection from splenomegaly and liver fibrosis resulting from intravascular hemolysis.

alpha(1)-Proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors, which function in maintaining homeostasis through regulation of numerous proteolytic processes. In laboratory mice (Mus musculus domesticus), alpha(1)-PI occurs in multiple isoforms encoded by a family of three to five genes that are polymorphic among inbred strains and that are located at the Serpina1 locus on chromosome 12. In the present study, we have characterized the alpha(1)-PI gene family of inbred mice in more detail. We show that mice express seven isoforms, all of which are encoded by genes that map to the Serpina1 locus. In addition, polymorphism at the locus is defined by three haplotypes (Serpina1(b), Serpina1(c), and Serpina1(l)) that differ with regard to both the number and identity of alpha(1)-PI genes. Finally, we present the complete sequence of an 84-kb region of Serpina1 containing a tandem repeat of two alpha(1)-PI genes.

Background: alpha(1)-Acid glycoprotein (alpha(1)-AGP) is an acute phase protein in most mammalian species whose concentration rises 2-5-fold during an acute phase reaction. Its serum concentration has often been used as a marker of disease, including inflammatory bowel disease (IBD). High alpha(1)-AGP levels were found to have a prognostic value for an increased risk of relapse in IBD., Aims: To investigate a possible role for increased serum levels of alpha(1)-AGP in the development of IBD., Methods: Dextran sodium sulphate (DSS) 2% was added to the drinking water of transgenic mice, overexpressing the rat alpha(1)-AGP gene, to induce acute colitis, thus mimicking the conditions of relapse. Clinical parameters, inflammatory parameters, and histological analyses on colon sections were performed., Results: Homozygous alpha(1)-AGP-transgenic mice started losing weight and showed rectal bleeding significantly earlier than heterozygous transgenic or wild-type mice. Survival time of homozygous transgenic mice was significantly shorter compared with heterozygous and wild-type mice. The higher susceptibility of homozygous alpha(1)-AGP-transgenic mice to DSS induced acute colitis was also reflected in higher local myeloperoxidase levels, higher inflammation scores of the colon, and higher systemic levels of interleukin 6 and serum amyloid P component. Local inflammatory parameters were also significantly different in heterozygous transgenic mice compared with wild-type mice, indicating a local dosage effect. In homozygous transgenic mice, significantly higher amounts of bacteria were found in organs but IgA levels were only slightly lower than those of control mice., Conclusion: Sufficiently high serum levels of alpha(1)-AGP result in a more aggressive development of acute colitis.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors that are involved in the regulation of a number of proteolytic processes. Alpha(1)-PI, like most serpins, functions by covalent binding to, and inhibition of, target proteinases. The interaction between alpha(1)-PI and its target is directed by the so-called reactive center loop (RCL), an approximately 20 residue domain that extends out from the body of the alpha(1)-PI polypeptide and determines the inhibitor's specificity. Mice express at least seven closely related alpha(1)-PI isoforms, encoded by a family of genes clustered at the Spi1 locus on chromosome 12. The amino acid sequence of the RCL region is hypervariable among alpha(1)-PIs, a phenomenon that has been attributed to high rates of evolution driven by positive Darwinian selection. This suggests that the various isoforms are functionally diverse. To test this notion, we have compared the proteinase specificities of individual alpha(1)-PIs from each of the two mouse species. As predicted from the positive Darwinian selection hypothesis, the various alpha(1)-PIs differ in their ability to form covalent complexes with serine proteinases, such as elastase, trypsin, chymotrypsin, and cathepsin G. In addition, they differ in their binding ability to proteinases in crude snake venoms. Importantly, the RCL region of the alpha(1)-PI polypeptide is the primary determinant of isoform-specific differences in proteinase recognition, indicating that hypervariability within this region drives the functional diversification of alpha(1)-PIs during evolution. The possible physiological benefits of alpha(1)-PI diversity are discussed.

Drug resistance gene therapy has the potential to protect against the myelosuppressive side effects of chemotherapy or to be used as a dominant in vivo selectable marker of genetically modified cells. Steady state kinetic studies have indicated the Escherichia coli thymidylate synthase (ecTS) is intrinsically more resistant to several TS-directed inhibitors as compared with the human enzyme, suggesting that ecTS is suitable for use as a drug-resistant marker. However, we found a disparity between the kinetic properties of ecTS and the degree of resistance conferred to cells transfected with the cDNA encoding this enzyme. It was determined that although ecTS is as stable as human TS (hTS) in transfected mammalian cells, ecTS is produced at only 40% the level of hTS, indicating poor translation of ecTS in eukaryotic cells. To circumvent this problem, the entire cDNA sequence of ecTS was synthesized by using codons optimized for expression in mammalian cells. In transfected Chinese hamster lung cells, expression of ecTS from the optimized construct, termed OPTecTS, is as efficient as hTS. Furthermore, cells transfected with the OPTecTS cDNA are significantly more resistant to the TS inhibitor raltitrexed as compared with transfected cells expressing similar levels of hTS. High-titer retroviral packaging cells were generated with OPTecTS and >80% of transduced mouse hematopoietic progenitor cells are resistant to raltitrexed, Thymitaq, and U89 at concentrations that eliminated colony growth of mock-transduced cells. The transgene was detectable by PCR in transduced bone marrow selected in U89 or raltitrexed, and expression of ecTS from the OPTecTS cDNA in bone marrow exhibited a catalytic rate constant comparable to that of purified recombinant ecTS. These data indicate that OPTecTS is a viable dominant selectable marker that can confer resistance to antifolates when introduced into cells.

Resistance to gram-negative infection can be induced by pretreating animals with several agents such as turpentine and interleukin (IL)-1. Because these agents are powerful inducers of acute phase proteins, we wondered whether these proteins, more particularly alpha(1)-acid glycoprotein (alpha(1)-AGP), are involved in nonspecific resistance to infection. Turpentine and IL-1 protect completely against a lethal challenge of Klebsiella pneumoniae when given 48 and 12-48 h before the challenge, respectively. alpha(1)-AGP induction in the serum reached peak values 48 h after turpentine and 12-48 h after IL-1 injection. Administration of alpha(1)-AGP, 2 h before a challenge of K. pneumoniae, significantly increased the survival. Numbers of bacteria cultured from blood and organs were significantly lower in mice pretreated with a protective dose of turpentine, IL-1, or alpha(1)-AGP. These data suggest that alpha(1)-AGP is a possible mediator in turpentine- or IL-1-induced protection because time points of maximal induction of alpha(1)-AGP by turpentine or IL-1 and of optimal protection by alpha(1)-AGP coincide. Transgenic overexpression of rat alpha(1)-AGP protected mice from a K. pneumoniae infection. Bacterial counts in blood and organs were significantly lower in transgenic mice, and only in control mice were large necrotic areas, apoptosis, and blood clots observed in the spleen. Our data suggest that alpha(1)-AGP prevents gram-negative infections and may be an essential component in nonspecific resistance to infection.

Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N5, N10-methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.

Previously, we identified evolutionarily derived changes in the pattern of nuclear factor binding to overlapping sites (termed A, B, and C, in proximal to distal order) within the promoter region of the murine D7Rp2e gene. In Mus domesticus, strong binding occurs at site B, with weak binding at sites A and C; in M. pahari, strong binding is observed at sites A and C, with weak binding at site B. The M. pahari-specific occupancy pattern is associated with repression of transcription. Presently, we have identified two interspecies changes, a single A --> G substitution within site B and a 7-bp indel within site C, that are primary determinants of the interspecies differences. These mutations alter both the pattern of binding site occupancy and the ability to repress transcription. The results are discussed in terms of a model involving a dynamic equilibrium between states of binding site occupancy, the nature of which can be modified during evolution.

Transcription of the murine D7Rp2e gene is highly variable among species of the genus Mus, indicating that extensive modifications in the gene's regulatory elements have occurred during evolution. Since promoter regions are well known to harbor cis-acting information that controls gene transcription, we compared the sequence and function of the D7Rp2e promoter in several Mus species, with the goal of understanding the molecular mechanisms underlying the interspecies variations in expression. Three overlapping binding sites for nuclear factors (sites A, B, and C in proximal to distal order) were identified about 300 bp upstream of the transcriptional start site. The sequences of these sites differ between the species Mus domesticus and M. pahari, which exhibit distinct D7Rp2e expression phenotypes. Site A binds a factor called RPBF-I; sites B and C bind a distinct factor that is termed RPBF-II and is likely a member of the NF-I family of transcription factors. DNase I footprinting experiments with the M. domesticus promoter show that binding of RPBF-II at site B is very strong, while binding of RPBF-I and RPBF-II at sites A and C, respectively, is weak; in contrast, with the M. pahari promoter, factor binding at sites A and C is strong, while that at site B is weak. These differences in patterns of binding-site occupancy derive from changes in the affinities of individual sites for their cognate nuclear factors. Transient transfection experiments indicate that the M. pahari binding pattern is capable of repressing transcription of a linked reporter. Such repression may contribute to the differences in D7Rp2e expression between the two species. We suggest that the species-specific footprinting patterns represent the existence of a dynamic equilibrium between two states of nuclear factor binding, the nature of which can be modified during evolution to result in new patterns of gene transcription.

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1-PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1-PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Although expression of the haptoglobin (HP) as an acute phase reactant is evolutionarily conserved among mammals, there are differences among species with regard to the hormones required for stimulation. Using primary hepatocyte cultures, we show that in Mus caroli, as in rat, IL-1 and IL-6 are stimulatory, whereas in M. domesticus, as in humans, IL-1 response is diminished. In vivo, an acute inflammatory process increases hepatic HP expression in both mouse species up to 30-fold but minimally affects the low level HP expression in the lung. To define the species-specific differences in regulation, we isolated the hormone-responsive elements of the HP gene from the Mus species, M. domesticus, M. caroli, and M. saxicola. Functional studies in transfected hepatoma cells revealed an exceptionally strong dexamethasone response for all three murine HP gene elements. The IL-6 response was less prominent than in rat or human. A modest response to IL-1 was observed in M. caroli and M. saxicola. A mouse-specific insertion of a polypurine sequence led to a binding site for the PEA3 transcription factor in the HP gene promoter of M. domesticus and M. saxicola, but not M. caroli. The specific regulatory effects of glucocorticoid receptor, C/EBP beta, and Ets proteins were documented by co-transfection.

Ornithine decarboxylase (ODC) is the first enzyme in the polyamine biosynthetic pathway. We have studied the evolutionary history of the mammalian ODC mRNA, focusing on the rate of accumulation of sequence divergence within specific subregions of the molecule. The phylogenetic relationships among the mRNAs from several mammalian species, including two mouse species, rat, hamster, and human, were determined based upon the numbers of synonymous substitutions in pairwise comparisons of mRNA coding regions. The separation times for the mRNAs were very similar to those for the corresponding species, suggesting that ODC is encoded by orthologous genes in the different species. Analysis of divergence patterns in four subregions, or domains, of the mRNA (the 5'-untranslated region, the coding region, and two domains of the 3'-untranslated region) showed that the domains have evolved in a noncoordinate fashion. Furthermore, evolution of each subregion has been episodic, with periods of both rapid and slow sequence divergence. We suggest that the episodic pattern of ODC mRNA evolution may indicate the existence of selection pressures that were exerted in a time- and domain-specific manner during mammalian speciation.

Two distinct members of the LINE-1 (L1) family in Peromyscus were characterized. The two clones, denoted L1Pm55 and L1Pm62, were 1.5 kb and 1.8 kb in length, respectively, and align to the identical region of the L1 sequence of Mus domesticus. Sequence similarity was on the order of 70% between L1Pm55 and L1Pm62, which approximates that between either Peromyscus sequence and Mus L1. L1Pm62 represents a more prevalent subfamily than L1Pm55. L1Pm62 exists in about 500 copies per haploid genome, while L1Pm55 exists in about 100 copies. The existence of major and minor subpopulations of L1 within Peromyscus is in contrast to murine rodents and higher primates, where L1 copy number is on the order of 20,000 to 100,000, and where levels of intraspecific divergence among L1 elements are typically less than 15-20%. Additional Peromyscus clones are similarly divergent from both L1Pm62 and L1Pm55, implying the existence of more than two distinct L1 subfamilies. The highly divergent L1 subfamilies in Peromyscus apparently have been evolving independently for more than 25 million years, preceding the divergence of cricetine and murine rodents. Investigations of the evolution of L1 within Peromyscus by restriction and Southern analysis was performed using species groups represented by the partially interfertile species pairs P. maniculatus-P. polionotus, P. leucopus-P. gossypinus, and P. truei-P. difficilis of the nominate subgenus and P. californicus of the Haplomylomys subgenus. Changes in L1 and species group taxonomic boundaries frequently coincided. The implications for phylogeny are discussed.

The expression of renal ornithine decarboxylase (ODC) is very different between the two murine species Mus domesticus and Mus pahari. The latter species contains a reduced level of ODC protein in the kidney, yet a normal concentration of ODC mRNA, indicating an alteration in translation, protein degradation, or both. We describe two lines of experimental evidence indicating that the efficiency of ODC mRNA translation is decreased in M. pahari. First, in vivo measurements showed that the rate of biosynthesis of the ODC polypeptide is reduced in M. pahari relative to M. domesticus. Second, polysome analyses indicated that, on average, the ODC mRNA of M. pahari binds fewer ribosomes. These effects, which are kidney-specific, lend support to the notion that renal ODC expression in the two species differs at the level of mRNA translation. A full-length cDNA copy of the ODC mRNA of M. pahari was isolated, and its sequence was compared with that of M. domesticus. In the 5'-untranslated region, which is likely to play an important role in controlling translation, the M. pahari transcript contains several single-base changes, as well as a 12-base deletion. These changes confer a distinct predicted secondary structure to this region of the mRNA and may be involved in reduced translation. We conclude that during evolution, modifications that alter the biosynthesis of specific proteins can occur at the translational level; these modifications exert their effects on translation in a transcript- and tissue-specific manner.