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Supplementary Figure 1 from The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1−/− mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF repressed cyclin D1 through a novel distal cis-element at −1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

Supplementary Figure 2 from The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1α, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1α, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.

Tire and road wear particles (TRWP) are a component of ambient particulate matter (PM) produced from the interaction of tires with the roadway. Inhalation of PM has been associated with cardiopulmonary morbidities and mortalities thought to stem from pulmonary inflammation. To determine whether TRWP may contribute to these events, the effects of subacute inhalation of TRWP were evaluated in rats. TRWP were collected at a road simulator laboratory, aerosolized, and used to expose male and female Sprague-Dawley rats (n = 10/treatment group) at ~10, 40, or 100 μg/m³ TRWP via nose-only inhalation for 6 h/day for 28 days. Particle size distribution of the aerosolized TRWP was found to be within the respirable range for rats. Toxicity was assessed following OECD guidelines (TG 412). No TRWP-related effects were observed on survival, clinical observations, body or organ weights, gross pathology, food consumption, immune system endpoints, serum chemistry, or biochemical markers of inflammation or cytotoxicity. Rare to few focal areas of subacute inflammatory cell infiltration associated with TWRP exposure were observed in the lungs of one mid and four high exposure animals, but not the low-exposure animals. These alterations were minimal, widely scattered and considered insufficient in extent or severity to have an impact on pulmonary function. Furthermore, it is expected that these focal lesions would remain limited and may undergo resolution without long-term or progressive pulmonary alterations. Therefore, from this study we identified a no-observable-adverse-effect-level (NOAEL) of 112 μg/m³ of TRWP in rats for future use in risk assessment of TRWP.

Alterations in insulin signaling as well as insulin action predispose to infertility as well as adverse pregnancy outcomes; however, little is known about the role of glucagon signaling in reproduction. The glucagon receptor knockout (Gcgr−/−) mouse created by our laboratory was used to define the role of glucagon signaling in maintaining normal reproduction. In this mouse model, lack of glucagon signaling did not alter the hypothalamic-pituitary-ovarian axis. Pregnant Gcgr−/−female mice displayed persistent hypoglycemia and hyperglucagonemia. Gcgr−/−pregnancies were associated with decreased fetal weight, increased late-gestation fetal demise, and significant abnormalities of placentation. Gcgr−/−placentas contained areas of extensive mineralization, fibrinoid necrosis, narrowing of the vascular channels, and a thickened interstitium associated with trophoblast hyperplasia. Absent glucagon signaling did not alter glycogen content in Gcgr−/−placentas but significantly downregulated genes that control growth, adrenergic signaling, vascularization, oxidative stress, and G protein-coupled receptors. Our data suggest that, similarly to insulin, glucagon action contributes to normal female reproductive function.

An oral sulfate salt solution (OSS), under development as a bowel cleansing agent for colonoscopy in humans, is studied in rats and dogs. In rats, amaximumpractical oral OSS dose (5 g/kg/d) is compared with an oral sodium phosphate (OSP) solution, both at about 7 times the clinical dose. OSS induces the intended effects of loose stools and diarrhea. In rats, higher urine sodium and potassium accompany higher clearance rates, considered adaptive to the osmotic load of OSS. OSS for 28 days is well tolerated in rats and dogs. In contrast, OSP causes increased mortality, reduced body weight and food consumption, severe kidney tubular degeneration, and calcium phosphate deposition in rats. These are accompanied by mineralization in the stomach and aorta, along with cardiac and hepatic degeneration and necrosis. The greater safety margin of OSS over OSP at similarmultiples of the clinical dose indicates its suitability for human use.

Two cats with intestinal malabsorption developed a hemorrhagic diathesis. Although unsubstantiated, the probable cause of bleeding was a chronic malabsorption of fat and the fat-soluble vitamin K. When treated with vitamin K1 per os, one cat's clotting times were only partially corrected. Since vitamin K1 is actively absorbed in the proximal small intestine, the incomplete response of this case to orally administered vitamin K1 was predictable. The infrequent occurrence of bleeding in animals with malabsorption is, in part, attributable to the ileal and colonic absorption of bacterially derived vitamin K2. For this reason, nonspecific use of antibiotics in these animals is contraindicated. Since long-chain, polyunsaturated fats impair vitamin K absorption, dietary fat given to animals with malabsorption should be restricted to medium- and short-chain, saturated fats. Vitamin K should be administered subcutaneously to these animals if prolonged clotting times or active bleeding is present, and routinely prior to surgery. Oral supplementation with vitamin K3, which is absorbed in the colon and less lipid soluble than vitamin K1, should be given to animals with malabsorption that are maintained as outpatients. Adequate dosage levels of vitamin K3, however, are yet to be established for the cat, and dose-dependent hemolytic anemia is a probable toxic manifestation.

We present a status report from the NCI Mouse Models of Human Cancers Consortium (MMHCC) Precancers Workshop held November 8 and 9, 2004. An expert panel, the Mouse Models Group (MMG) evaluated the status of mouse models of precancer emphasizing genetically engineered mouse models, especially of lining epithelium and their utilitarian value to human carcinogenesis. An outline of the background for the panel’s considerations is provided with examples of past and current precancerous lesions in mice. The experimental use of oncogenic viruses and chemical carcinogens in mice led to operational definitions of initiation, promotion, and preneoplasia Preneoplastic and precancerous lesions are found in these models. In this precancer concept, most preneoplastic lesions are considered as potentially precancerous or at least an earlier stage in cancer development than typical pre-invasive epithelial lesions, which are often seen in these mouse models. Genetically engineered mice, used to test the oncogenicity of individual genes, develop precancers that are initiated by defined molecular and histopathologic changes. The mouse can be used to isolate and study precancers in detail, thereby providing a level of biological understanding not readily available in clinical disease. These studies suggest that genetically engineered mice are very useful preclinical models for chemoprevention and therapy.

Objectives To perform a comparison to determine which of two methods of partial urethral ligation produces the most consistent outcome and fewest side effects. Such a study has not been previously reported. Partial urethral ligation is a means of causing reproducible bladder outlet obstruction. In the male rat model, partial urethral obstruction can be performed either by perineal incision and bulbous urethral ligation or retropubic incision and midprostatic obstruction. Methods Fifteen male Sprague-Dawley rats were studied. Five were selected for bulbous urethral obstruction through a perineal incision, five for midprostatic obstruction using a retropubic approach, and five for a sham operation through a perineal incision. Results The operative time was shorter and morbidity lower with the perineal approach compared with the retropubic approach. Inflammation or infection, or both, were seen in the prostate, bladder, proximal urethra, ureters, and kidneys in the rats in which a midprostatic obstruction was performed. The proximal urethra and prostate were mildly inflamed in those rats that underwent bulbous obstruction. Sham-operated rats exhibited mild prostatitis only. Conclusions The perineal approach to the bulbous urethra is the method of choice for creating a partial urethral obstruction model of bladder outlet obstruction in the male rat.

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid β-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARγ and PPARδ agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARγ agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARδ agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARγ agonist expressed estrogen receptor-α in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARδ agonist exhibited increased PPARδ levels and activated 3-phosphoinositide–dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARδ activation. These results indicate that PPARδ and PPARγ agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.

Mouse mammary tumors arising during medroxyprogesterone-DMBA-mediated mammary carcinogenesis comprised three distinct phenotypes: adenocarcinoma, squamous cell carcinoma, and myoepithelial carcinoma. The molecular signature for each of the three tumor subsets was characterized by gene microarray analysis, and three distinct sets of gene expression profiles were obtained that were corroborated in part by quantitative RT-PCR and immunohistochemistry. These results suggest that this carcinogenesis and gene expression model will be useful for rapidly assessing the histopathological differences arising in mammary carcinogenesis and the effects of tumor promoting or chemoprevention agents.

Oncogenic signals elevate expression of Id2 in multiple tumor types. When deregulated, Id2 inactivates the tumor suppressor proteins retinoblastoma, p107, and p130. Here, we report a novel and unexpected tumor inhibitory function of Id2 in the intestinal epithelium. First, genetic ablation of Id2 in the mouse prevents differentiation and cell cycle arrest of enterocytes at the time of formation of the crypt-villus unit. Later, these developmental abnormalities evolve toward neoplastic transformation with complete penetrance. Id2-null tumors contain severe dysplastic and metaplastic lesions and express aberrant amounts of β-catenin. Thus, our data are the first to establish a direct requirement of basic helix-loop-helix inhibitors in driving differentiation and define an unexpected role for the retinoblastoma-binding protein Id2 in preventing tumor formation.

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1−/− mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF repressed cyclin D1 through a novel distal cis-element at −1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The Δ leuD Δ panCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Caveolae are flask-shaped invaginations of the plasma membrane formed by the oligomerization of caveolins. Because only smooth muscle contains all caveolin (Cav) family members (Cav-1, 2 and 3), we examined the contribution of each caveolin to urogenital smooth muscle structure/function.WT, Cav-1, 2, 3 and -1/3 knockout (KO) mouse bladders were characterized by Western blot, co-immunoprecipitation, immunofluorescence microscopy, electron microscopy, histochemistry and pharmacological techniques. Cystometric analysis was performed in conscious, freely moving mice. Other urogenital organs were investigated by histological analysis.The loss of bladder Cav-1 results in a marked decrease in Cav-2 but not Cav-3 expression. Ablation of Cav-3 fails to alter Cav-1 or Cav-2 expression. Deletion of Cav-1 results in the almost complete loss of caveolae, while Cav-2 KO and Cav-3 KO mouse smooth muscle showed a normal number of caveolae. The loss of Cav-1 generated caveolae led to significant urogenital changes in male mice (most marked by 12 months of age), namely 1) bladder weight-to-body weight ratios were increased, 2) the bladder smooth muscle layer was thickened, 3) the bladders had increased baseline, threshold and spontaneous pressures, 4) bladder strips showed a decreased contractile response to carbachol and KCl, and 5) these smooth muscle changes were accompanied by marked fluid accumulation in the prostate and seminal vesicles, with intracellular vacuolization in the kidneys. As such, male Cav-1 KO mice may be a useful animal model for studying LUTD (lower urinary tract dysfunction) that is so prevalent in aging male patients.The loss of Cav-1 and, thus, of most smooth muscle cell caveolae results in significant bladder dysfunction and urogenital organ changes in aged male mice.

Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

Caveolin-1 (Cav-1) is the principal structural protein of caveolae membranes that are found in most cells types, including mammary epithelial cells. Recently, we mapped the human CAV1 gene to a suspected tumor suppressor locus (7q31.1/D7S522) that is deleted in a variety of human cancers, as well as mammary tumors. In addition, the CAV1 gene is mutated (P132L) in up to ∼16% of human breast cancers. The mechanism by which deletion or mutation of the Cav-1 gene contributes to mammary tumorigenesis remains unknown. To understand the role of the Cav-1 (P132L) mutation in the pathogenesis of human breast cancers, we generated the same mutation in wild-type (WT) Cav-1 and studied its behavior in cultured cells. Interestingly, the P132L mutation leads to formation of misfolded Cav-1 oligomers that are retained within the Golgi complex and are not targeted to caveolae or the plasma membrane. To examine whether the Cav-1 (P132L) mutant behaves in a dominant-negative manner, we next co-transfected cells with Cav-1 (P132L) and WT Cav-1, and evaluated their caveolar targeting. Our results indicate that Cav-1 (P132L) behaves in a dominant-negative manner, causing the mislocalization and intracellular retention of WT Cav-1. Virtually identical results were obtained when Cav-1 (P132L) was stably expressed at physiological levels in a nontransformed human mammary epithelial cell line (hTERT-HME1). These data provide a molecular explanation for why only a single mutated CAV1 allele is found in patients with breast cancer. Thus, we next investigated if functional inactivation of Cav-1 gene expression leads to mammary tumorigenesis in vivo. For this purpose, we performed mammary gland analysis on Cav-1-deficient mice (−/−) that harbor a targeted disruption of the Cav-1 gene (a null mutation). Interestingly, we show that inactivation of Cav-1 gene expression leads to mammary epithelial cell hyperplasia, even in 6-week-old virgin female mice. These data clearly implicate loss of functional Cav-1 in the pathogenesis of mammary epithelial cell hyperplasia, and suggest that Cav-1-null mice represent a novel animal model to study premalignant mammary disease.

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (−/−) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (−/−) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

With the advent of HIV and the widespread emergence of drug-resistant strains of Mycobacterium tuberculosis, newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is an ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo. Attenuated M. tuberculosis vaccine candidates have been constructed by deleting genes required for growth in mice. These candidate vaccines did not elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of M. tuberculosis defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer (250 days) than mice infected with either bacille Calmette-Guerin (BCG) vaccine or virulent M. tuberculosis (77 and 35 days, respectively). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent M. tuberculosis, which was comparable with that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of M. tuberculosis without reducing its immunogenicity in vaccinated mice.

The neu (c- erbB-2, HER2 ) proto-oncogene encodes a receptor tyrosine kinase that is a member of an important growth factor receptor family which includes the epidermal growth factor receptor (EGFR, ErbB1), ErbB3 and ErbB4. The neu is found over-expressed in 20–30% of human breast tumors [1] . The c- erbB-2 is sufficient for the induction of mammary tumorigenesis in transgenic mice and the pathology of these mammary tumors strongly resembles human breast cancer. Murine transgenic models engineered to recapitulate human breast cancer provide an excellent and straightforward approach to dissect the molecular mechanisms governing the onset and progression of this disease. The molecular mechanisms by which ErbB-2 transforms cells involves direct effects on components of the cell-cycle regulatory apparatus. Recent studies have demonstrated a key role for components of the cell-cycle, in particular cyclin D1 and p27Kip1 (p27) in the onset and progression of ErbB-2-induced murine mammary tumorigenesis. Such studies have provided further impetus to therapeutics targeting these cell-cycle proteins.

F lap e ndo n uclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1 . Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli ( Apc ) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.

The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.

In human breast carcinomas, overexpression of the macrophage colony–stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1op) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1op/Csf1op and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.

Genomic integrity is continuously threatened by endogenous and exogenous agents that damage DNA and have immediate and long-term effects on cellular functioning. Replication of damaged DNA can cause mutations that may ultimately lead to carcinogenic events or, when occurring in germ cells, to inborn defects. Immediate effects of DNA damage include a blockage to transcription which can result in cell death by apoptosis (22). To minimize the harmful effects of DNA damage, nature has equipped cells with a sophisticated network of DNA repair mechanisms. Nucleotide excision repair (NER) is a cut-and-paste repair mechanism for the removal of a variety of bulky DNA lesions, such as chemical adducts and UV-induced photolesions (reviewed in references 11 and 16). The NER apparatus utilizes approximately 30 gene products to (i) recognize DNA damage, (ii) introduce single-stranded nicks 5′ and 3′ to the lesion, resulting in the removal of a single-stranded oligonucleotide containing the lesion, and (iii) restore the integrity of the double helix by gap-filling DNA synthesis (using the undamaged DNA strand as a template) and strand ligation (16). NER can be divided into two subpathways that differ in the molecular mechanism of lesion recognition. The global genome repair machinery (ggNER), in which damage recognition is performed by the XPC-HR23B complex (54), can remove lesions located anywhere in the genome. However, some types of lesions are poorly recognized by the ggNER apparatus and therefore are inefficiently repaired. Such lesions, when present in the template strand of active genes, interfere with transcription and activate the transcription-coupled repair subpathway of NER (tcNER). In the tcNER reaction, stalling of RNA polymerase II at bulky lesions serves as the damage sensor and subsequently recruits the remainder of the NER apparatus (37, 38, 55, 56). The importance of NER for genome preservation is illustrated by rare autosomal recessive disorders like xeroderma pigmentosum (XP), which is made up of seven complementation groups (XPA through XPG), and Cockayne syndrome (CS), which is made up of two complementation groups (CSA and CSB) (5). Both disorders are characterized by hypersensitivity to solar (UV) light. XP individuals demonstrate an increased rate of sunlight-induced skin cancer as well as carcinogen-induced internal tumors (5, 34). Except for XPC and XPE, where only the ggNER pathway is affected, XP is associated with a defect in both ggNER and tcNER. Individuals with CS exhibit small stature, intellectual impairment, cachexia, and sun sensitivity but no increased incidence of cancer (5, 15, 24). Cells from CS patients have been shown to be deficient in tcNER only (11, 16, 26, 57). An important question is why CS patients, despite their DNA repair deficiency, have not been reported to develop cancer of the skin or internal organs. A possible explanation is that CS cells can still perform ggNER, which, even though some types of lesions may be repaired at lower rates, could prevent accumulation of mutagenic lesions (15). Alternatively, since CSA and CSB cells cannot appropriately clear RNA polymerase II arrested at intragenic DNA lesions (39) and since stalled RNA polymerase II has been shown to promote intracellular accumulation of p53 and apoptosis (41, 53, 62), it has been suggested that in CS patients precancerous cells are more efficiently eliminated by apoptosis than they are in healthy persons (24). Among the growing number of mouse mutants with engineered deletions of genes involved in cellular responses to DNA damage are mouse models for XPA (18, 42), CSA (21), and CSB (59). Totally NER-deficient XPA−/− mice resemble human XPA patients in their UV-induced skin cancer predisposition (18, 42) and also show elevated frequencies of skin and internal tumors after exposure to chemical carcinogens (17, 18). In contrast to human CS, CSB−/− mice demonstrate an elevated incidence of both UV- and chemically induced skin cancer (59). This difference between CSB patients and mice might be due to the lack of expression of the p53-inducible p48 gene, required for ggNER of UV-induced cyclobutane dimers (and presumably other carcinogen-induced DNA lesions) (27). Since removal of these types of lesions in rodents almost totally depends on tcNER, the CSB defect may have a more dramatic effect in mice than in humans. Alternatively, since UV-induced skin tumor formation in CSB mice requires a longer latency time than that required by totally NER-deficient XPA mice (3, 4), CSB patients may simply not get old enough (average life span, 12.5 years) to develop tumors (43). Totally NER-deficient XPA mice, like human XP patients, develop spontaneous internal tumors upon aging (17). With the observation that, at least in mice, a specific tcNER defect predisposes to UV- and chemically induced skin cancer, the question of whether CS is also associated with increased sensitivity to spontaneous tumor formation arises. Mice with a homozygous disruption of the Ink4a/ARF locus have proven to be a particularly useful model system for studying factors (such as Ras and telomerase) contributing to neoplasia (9, 23, 48, 49). A great deal of evidence indicates that deletion of this region predisposes to cancer development (reviewed in references 8, 50, and 51). Humans with lesions in this genetic locus have an elevated incidence of melanoma, pancreatic carcinoma, and other neoplasms. Mice with homozygous disruption of the Ink4a/ARF locus are predisposed to developing spontaneous lymphomas and fibrosarcomas (49). The Ink4a gene product, p16Ink4a, helps regulate cellular proliferation by inhibiting cyclin-dependent kinases 4 and 6. The ARF gene product, p19ARF, is encoded by a gene overlapping that of the Ink4a gene but utilizing an alternative reading frame (ARF). p19ARF potentiates the function of p53 by antagonizing mdm2 function (52). If left unimpeded, mdm2 inhibits p53 function both by binding and by blocking p53's transcriptional activation domain as well as by catalyzing p53's ubiquitination, thus leading to its proteasomal degradation (32). Cancer-predisposed Ink4a/ARF−/− mice could therefore be used to investigate the role of the CSB gene product in spontaneous tumorigenesis. In the present study we have generated CSB−/− Ink4a/ARF−/− mice to study the effect of a CSB deficiency on spontaneous tumor formation. Surprisingly, since DNA repair genes are usually considered tumor suppressor genes, we found that inactivation of the CSB gene reduced spontaneous tumor formation in Ink4a/ARF−/− mice. Comparison of CSB−/− Ink4a/ARF−/−, CSB−/−, Ink4a/ARF−/−, and wild-type (WT) mouse embryo fibroblasts (MEFs) for their colony formation rates after low density seeding, H-Ras oncogene-mediated transformation rates, proliferation rates, and mRNA transcription rates revealed that at the cellular level, the CSB gene deficiency diminished neoplastic potential. Moreover, we show that the CSB gene deficiency sensitizes Ink4a/ARF−/− MEFs to UV-induced accumulation of p53 and apoptosis. Similarly, the CSB defect is shown to reduce the neoplastic potential of p53−/− MEFs. The implications of a CSB deficiency for tumorigenesis are discussed.

Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of “cat eye” and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.

Attenuated mutants ofMycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph ofM. tuberculosiswas created by allelic exchange, replacing wild-typeleuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was −11. In contrast to wild-typeM. tuberculosis, the ΔleuDmutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-typeleuDabolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuDmutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis, were immunized with the ΔleuDmutant and subsequently challenged with virulentM. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovisBCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuDmutant ofM. tuberculosisexhibited enhanced and statistically equivalent survival curves. However, theleuDauxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection againstM. tuberculosischallenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosisshould persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

A necessary role for cytotoxic T lymphocytes in protection againstMycobacterium tuberculosis(MTB) has been suggested by studies of the β2-microglobulin-deficient mouse, which is unable to present antigens through MHC class I and class I-like molecules and invariably succumbs early after infection. To identify the relative contributions of distinct putative MHC class I-dependent cell populations in protection against tuberculosis, we compared a variety of gene-disrupted mouse strains for susceptibility to MTB infection. Among the strains tested, the most susceptible mice, as measured by survival time and bacterial loads, were the β2-microglobulin−/−, followed by transporter associated with antigen processing deficient (TAP1−/−), CD8α−/−, perforin−/−, and CD1d−/−mice. These findings indicated that (i) CD8+T cells contribute to protection against MTB, and their protective activity is only partially dependent on perforin; (ii) β2-microglobulin-dependent T cell populations distinct from CD8+T cells also contribute to anti-MTB immunity; and (iii) protective immune mechanisms are predominantly TAP-dependent, although TAP-independent mechanisms also contribute to protection. Because CD1d-deficient animals were fully resistant to MTB, other TAP-independent mechanisms must contribute to protection. We suggest here that both classical and nonclassical MHC class I-restricted T cells, distinct from CD1d-restricted cells, may be involved in protective immune responses against tuberculosis.

Bcl-2 is an anti-apoptotic and anti-proliferative protein over-expressed in several different human cancers including breast. Gain of Bcl-2 function in mammary epithelial cells was superimposed on the WAP-TAg transgenic mouse model of breast cancer progression to determine its effect on epithelial cell survival and proliferation at three key stages in oncogenesis: the initial proliferative process, hyperplasia, and cancer. During the initial proliferative process, Bcl-2 strongly inhibited both apoptosis and mitotic activity. However as tumorigenesis progressed to hyperplasia and adenocarcinoma, the inhibitory effects on mitotic activity were lost. In contrast, anti-apoptotic activity persisted in both hyperplasias and adenocarcinomas. These results demonstrate that the inhibitory effect of Bcl-2 on epithelial cell proliferation and apoptosis can separate during cancer progression. In this model, retention of anti-apoptotic activity with loss of anti-proliferative action resulted in earlier tumor presentation.

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1 , in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1 -deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27 KIP1 abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin–radixin–moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1γ. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo .

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.

Obstacles to the expansion of cells with proliferative potential include the induction of cell death, telomere-based senescence, and the pRb and p53 tumor suppressors. Not infrequently, the molecular pathways regulating oncogenesis recapitulate aberrations of processes governing embryogenesis. The genetic network, consisting of the dachshund (dac), eyes absent (eya), eyeless, and sine oculis (so) genes, regulates cell fate determination in metazoans, with dac serving as a cointegrator through a So DNA-binding factor. Here, DACH1 inhibited oncogene-mediated breast oncogenesis, blocking breast cancer epithelial cell DNA synthesis, colony formation, growth in Matrigel, and tumor growth in mice. Genetic deletion studies demonstrated a requirement for cyclin D1 in DACH1-mediated inhibition of DNA synthesis. DACH1 repressed cyclin D1 through a novel mechanism via a c-Jun DNA-binding partner, requiring the DACH1 alpha-helical DS domain which recruits corepressors to the local chromatin. Analysis of over 2,000 patients demonstrated increased nuclear DACH1 expression correlated inversely with cellular mitosis and predicted improved breast cancer patient survival. The cell fate determination factor, DACH1, arrests breast tumor proliferation and growth in vivo providing a new mechanistic and potential therapeutic insight into this common disease.

The inhibitor of differentiation Id2 is a target of the retinoblastoma (Rb) protein during mouse embryogenesis. In Rb(+/-) mice, LOH at the wild-type Rb allele initiates pituitary adenocarcinoma, a tumor derived from embryonic melanotropes. Here we identify a critical role for Id2 in initiation, growth, and angiogenesis of pituitary tumors from Rb(+/-) mice. We show that proliferation and differentiation are intimately coupled in Rb(+/-) pituitary cells before tumor initiation. In Id2-null pituitaries, premature activation of basic helix-loop-helix-mediated transcription and expression of the cdk inhibitor p27(Kip1) impairs the proliferation of melanotropes and tumor initiation. Without Id2, Rb(+/-) mice have fewer early tumor lesions and a markedly decreased proliferation rate of the tumor foci. Expression of Id2 by pituitary tumor cells promotes growth and angiogenesis by functioning as a master regulator of vascular endothelial growth factor (VEGF). In human neuroblastoma, the N-Myc-driven expression of Id2 is sufficient and necessary for expression of VEGF. These results establish that aberrant Id2 activity directs initiation and progression of embryonal cancer.

We report the safety and immunogenicity of a double lysine and pantothenate auxotroph ofMycobacterium tuberculosisin mice. The ΔlysAΔpanCDmutant is completely attenuated in immunocompromised SCID and gamma interferon knockout mice yet induces short-term and long-term protection in immunocompetent and CD4-deficient mice following single-dose subcutaneous vaccination.

Constitutive beta-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (ApcMin) develop intestinal adenomas. Here, the crossing of ApcMin with cyclin D1-/- mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of ApcMin/cyclin D1+/- mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.

The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.

Adiponectin is a plasma protein expressed exclusively in adipose tissue. Adiponectin levels are linked to insulin sensitivity, but a direct effect of chronically elevated adiponectin on improved insulin sensitivity has not yet been demonstrated. We identified a dominant mutation in the collagenous domain of adiponectin that elevated circulating adiponectin values in mice by 3-fold. Adiponectinemia raised lipid clearance and lipoprotein lipase activity, and suppressed insulin-mediated endogenous glucose production. The induction of adiponectin during puberty and the sexual dimorphism in adult adiponectin values were preserved in these transgenic animals. As a result of elevated adiponectin, serum PRL values and brown adipose mass both increased. The effects on carbohydrate and lipid metabolism were associated with elevated phosphorylation of 5'-AMP-activated protein kinase in liver and elevated expression of peroxisomal proliferator-activated receptor gamma2, caveolin-1, and mitochondrial markers in white adipose tissue. These studies strongly suggest that increasing endogenous adiponectin levels has direct effects on insulin sensitivity and may induce similar physiological responses as prolonged treatment with peroxisomal proliferator-activated receptor gamma agonists.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette–Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region ( RD1 ), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting Δ RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis Δ RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the Δ RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 ( cfp-10 esat-6 ) operon of RD1 , also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette–Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs inor =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.

Exonuclease 1 (Exo1) is a 5′–3′ exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated anExo1mutant mouse line. Analysis ofExo1−/−cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5′ and 3′ nick-directed repair. The repair defect inExo1−/−cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at theHprtlocus.Exo1−/−animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition,Exo1−/−male and female mice were sterile because of a meiotic defect. Meiosis inExo1−/−animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.

A number of cancer chemotherapeutic drugs designed to have cytotoxic actions on tumor cells have recently been shown to also have antiangiogenic activities. Endothelial cell migration and proliferation are key components of tumor angiogenesis, and agents that target the microtubule cytoskeleton can interfere with these processes. In this study, the effect on endothelial cell functions of the microtubule-stabilizing drugs Taxotere and Taxol were evaluated in three in vitro assays: a chemokinetic migration assay, an angiogenesis factor-mediated chemotactic migration assay, and a three-dimensional Matrigel tubule formation assay, using rat fat pad endothelial cells (RFPECs) and/or human umbilical vein endothelial cells (HUVECs). Taxotere was active in all three assays at concentrations that were not cytotoxic and did not inhibit endothelial cell proliferation. In the RFPEC chemokinetic migration and in vitro tubule formation assays, the IC50 values were approximately 10(-9) M for both Taxotere and Taxol. HUVEC migration, however, was more sensitive to Taxotere, with an observed IC50 of 10(-12) M in a chemokinetic assay. In a Boyden chamber assay, HUVEC chemotaxis stimulated by either of two angiogenic factors, thymidine phosphorylase or vascular endothelial growth factor, was inhibited by Taxotere with an IC50 of 10(-11) M and was ablated at 10(-9) M. Taxotere was also up to 1000-fold more potent than Taxol in inhibiting either chemokinetic or chemotactic migration. When the microtubule cytoskeleton was visualized using immunofluorescence staining of alpha-tubulin, there were no gross morphological changes observed in HUVECs or RFPECs treated with Taxotere at concentrations that inhibited endothelial cell migration but not proliferation. The effects of Taxotere on migration were associated with a reduction in the reorientation of the cell's centrosome, at concentrations that did not affect gross microtubule morphology or proliferation. Reorientation of the centrosome, which acts as the microtubule organizing center, in the intended direction of movement is a critical early step in the stabilization of directed cell migration. These data indicate that endothelial cell migration correlates more closely with changes in microtubule plasticity than with microtubule gross structure. The antiangiogenic activity of Taxotere in vivo was assessed in a Matrigel plug assay. In this assay, the angiogenic response to fibroblast growth factor 2 was inhibited in vivo by Taxotere with an ID50 of 5.4 mg/kg when injected twice weekly over a 14-day period, and angiogenesis was completely blocked in mice that received 10 mg/kg Taxotere. The in vivo data further suggested that Taxotere had selectivity for endothelial cell migration and/or microvessel formation because infiltration of inflammatory cells into the Matrigel plug was much less sensitive to inhibition by Taxotere. In conclusion, Taxotere is a potent and potentially specific inhibitor of endothelial cell migration in vitro and angiogenesis in vitro and in vivo.

Caveolae organelles and caveolin-1 protein expression are most abundant in adipocytes and endothelial cells. Our initial report on mice lacking caveolin-1 (Cav-1) demonstrated a loss of caveolae and perturbations in endothelial cell function. More recently, however, observation of the Cav-1-deficient cohorts into old age revealed significantly lower body weights, as compared with wild-type controls. These results suggest that Cav-1 null mice may have problems with lipid metabolism and/or adipocyte functioning. To test this hypothesis directly, we placed a cohort of wild-type and Cav-1 null mice on a high fat diet. Interestingly, despite being hyperphagic, Cav-1 null mice show overt resistance to diet-induced obesity. As predicted, adipocytes from Cav-1 null null mice lack caveolae membranes. Early on, a lack of caveolin-1 selectively affects only the female mammary gland fat pad and results in a near complete ablation of the hypo-dermal fat layer. There are also indications of generalized adipose tissue pathology. With increasing age, a systemic decompensation in lipid accumulation occurs resulting in dramatically smaller fat pads, histologically reduced adipocyte cell diameter, and a poorly differentiated/hypercellular white adipose parenchyma. To gain mechanistic insights into this phenotype, we show that, although serum insulin, glucose, and cholesterol levels are entirely normal, Cav-1 null mice have severely elevated triglyceride and free fatty acid levels, especially in the post-prandial state. However, this build-up of triglyceride-rich chylomicrons/very low density lipoproteins is not due to perturbed lipoprotein lipase activity, a major culprit of isolated hypertriglyceridemia. The lean body phenotype and metabolic defects observed in Cav-1 null mice are consistent with the previously proposed functions of caveolin-1 and caveolae in adipocytes. Our results show for the first time a clear role for caveolins in systemic lipid homeostasis in vivo and place caveolin-1/caveolae as major factors in hyperlipidemias and obesity.

DNA in the nuclei of all eukaryotic cells is packaged into repeating units of nucleosomes that form the basic unit of chromatin. Each nucleosome consists of an octamer core containing two molecules of each of the core histones, H2a, H2b, H3, and H4. H1 linker histones bind to the nucleosome core particle and the linker DNA between nucleosomes to facilitate further compaction of chromatin into a 30-nm fiber. Recent studies have shown that the chromatin complex, especially the nucleosome and its modifications, can have a profound influence on transcription (reviewed in references 9 and 26). Although histones are highly conserved proteins, multicellular organisms contain a variety of subtypes exhibiting significant sequence divergence. Among the histone classes, the H1 linker histones are the most divergent group. In mammals, there are at least eight H1 subtypes, including the somatic H1s, H1a to H1e, germ cell-specific H1t and H1oo, and replacement linker histone H10 (11, 24). These subtypes exhibit distinct patterns of expression during differentiation and development (12, 24). The significance of the diversity present within the H1 family is not understood. The genes for H1a through H1e and H1t are tightly linked on mouse chromosome 13 (25). The H10 gene is located on mouse chromosome 15 (2). H10 is the smallest and most divergent member of the H1 family (27). H10 accumulates in quiescent cells and during terminal differentiation and terminal cell division, reaching levels as high as 30% of the total H1 in certain tissues, such as adult liver. Despite the unique properties and developmental regulation of H10, previous studies in our laboratory showed that mice develop normally without H10 (23). Analysis of chromatin from H10-null mice indicated that the level of the somatic H1s, especially H1c, H1d, and H1e, was increased so as to maintain a normal ratio of H1 to nucleosomes in H10-deficient chromatin. In certain tissues, such as adult liver, H1c, H1d, and H1e accounted for 95% of the remaining H1, suggesting that these subtypes are responsible for compensating for loss of H10. The present study was undertaken with the following two experimental objectives: first, to determine whether or not any one of several H1 subtypes is essential for mouse development; second, to determine whether H1c, H1d, or H1e is responsible for compensating for the loss of H10 in H10−/− mice. To achieve the first goal, we generated null mutations in each of the three somatic H1 genes by homologous recombination in embryonic stem (ES) cells and then produced mice lacking each of these individual subtypes. To achieve the second goal, we bred each of these three H1 knockout mice to H10 null mice and ultimately produced H1c/H10, H1d/H10, and H1e/H10 double-knockout mice.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1–nullCsf1op/Csf1opmice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects ofCsf1op/Csf1opmice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed β-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

The reduced folate carrier (RFC1) is an important route by which the major blood folate, 5-methyltetrahydrofolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1(-)/- embryos examined at E18.5 by supplementation of pregnant RFC1(+/-) dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. These RFC1(-)/- mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.