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Perspective on this Article from Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

High levels of the critical p53 inhibitor Mdm4 is common in tumors that retain a wild-type p53 allele, suggesting that Mdm4 overexpression is an important mechanism for p53 inactivation during tumorigenesis. To test this hypothesis in vivo, we generated transgenic mice with widespread expression of Mdm4. Two independent lines of transgenic mice, Mdm4Tg1 and Mdm4Tg15, developed spontaneous tumors, the most prevalent of which were sarcomas. To determine whether overexpression of Mdm4 also cooperated with p53 heterozygosity to induce tumorigenesis, we generated Mdm4Tg1 p53+/− mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53+/− or Mdm4Tg1 mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4Tg1 p53+/− tumors. Mdm4 is thus a bona fide oncogene in vivo and cooperates with p53 heterozygosity to drive tumorigenesis. These Mdm4 mice will be invaluable for in vivo drug studies of Mdm4 inhibitors. Cancer Res; 70(18); 7148–54. ©2010 AACR.

Supplementary Methods, Figure Legends 1-3 from Overexpression of the Low Molecular Weight Cyclin E in Transgenic Mice Induces Metastatic Mammary Carcinomas through the Disruption of the ARF-p53 Pathway

Supplementary Figure 3 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Figure 1 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Methods, Figure Legends 1-3 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Figures 1-3, Table 1 from Overexpression of the Low Molecular Weight Cyclin E in Transgenic Mice Induces Metastatic Mammary Carcinomas through the Disruption of the ARF-p53 Pathway

Supplementary Table 1 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Figure 2 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor–bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy. [Cancer Res 2007;67(15):7212–22]

Background The Nurse Assistant App (NAA) was developed to improve the quality of antenatal care in Magu District Tanzania by providing digital assistance to healthcare workers. The NAA guides healthcare workers step by step through all necessary antenatal care interventions and provides tailored advice and suggestions for follow-up actions. We used the Intervention Mapping framework to retrospectively evaluate the development of the NAA. Methods Desk research into the content, evidence base, and development of the NAA was performed combined with semi-structured interviews with two initial developers to fill in a planning tool consisting of 40 criteria for systematic evaluation of the data. Results Findings showed that the NAA was developed based on existing literature on antenatal care guidelines as well as the knowledge and experience of a broad variety of stakeholders and that five of the six steps of Intervention Mapping were performed. However, the development of the NAA lacked a theoretical foundation on behaviour change models. Future research will focus on the experiences of healthcare workers providing antenatal care using the NAA. Conclusions The NAA was developed in collaboration with relevant stakeholders and took practical matters as well as evidence from existing literature into account. However, we recommend that the responsibility and ownership of the NAA should be shared with the local government to increase the potential and sustainability of the NAA. Key messages The NAA was developed to improve the quality of antenatal care in rural Tanzania targeting healthcare workers. The current evaluation informs future digital health tool developers in sub-Saharan Africa about elements to take into account during the development phase such as systematic mapping of the process.

Background To reduce maternal mortality in rural Tanzania, the need to improve antenatal care remains an urgent priority. Digital health tools are seen as a promising instrument to increase the quality of healthcare in developing countries, but it remains unclear to what extent they are perceived as beneficial during the antenatal care visit. Therefore, the aim of this research was to identify factors that could improve the quality of antenatal care in rural Tanzania, and whether digital health tools would change healthcare workers' and pregnant women's perceptions of quality of care. Methods Semi-structured in-depth interviews were conducted with sixteen healthcare workers and nineteen pregnant women, recruited from various health facilities in Magu District, Tanzania. Results Both healthcare workers and pregnant women possess a positive attitude towards antenatal care and acknowledge its importance. However, they also expressed a need for (structural) improvements in antenatal care services, particularly increased availability of diagnostic tests, and increased performance and motivation of healthcare workers. In addition, healthcare workers indicated a need for more training and better supervision. While both groups expected quality of care to increase should a digital health tool be implemented, they placed more focus on investing in health infrastructure, like the availability of medication and other materials, than investments in digital health. Conclusions Infrastructural improvements, including better communication between healthcare workers and pregnant women, as well as training opportunities and supportive leadership, are necessary before a digital health tool can be expected to increase the quality of antenatal care. Key messages Both healthcare workers and pregnant women acknowledged the need for higher quality antenatal care. Both groups suggested a number of infrastructural improvements, among which the use of a digital health tool.

Background & Aim Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in patients with high-risk disease, and traditional therapies often cause significant permanent morbidity. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy because they recognize malignant cells without the need for a known target, they are readily available from multiple banks, and they expand to significant numbers. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment, such as Transforming Growth Factor β (TGF-β). Methods, Results & Conclusion To overcome the detrimental effects of TGF-β, we modified CB-derived NK cells to express a dominant negative TGF-β receptor II (DNRII) by retroviral transduction and evaluated their ability to kill a TGF-β-secreting MB cell line (Daoy). We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence ofmedulloblastoma-conditioned, TGF-β-richmedia (decreasing to 14.9±2.1% killing at an E:T ratio of 5:1 from 25.1±2.9%, n=10, p Hence, CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation as a novel therapeutic for the treatment of patients with high-risk medulloblastoma.

Wind erosion is a threat to the sustainability and productivity of soils that takes place at local, regional, and global scales. Current estimates of the cost of wind erosion have not included the costs associated with the loss of soil biodiversity and reduced ecosystem functions. Microorganisms carried in dust are responsible for numerous critical ecosystem processes including biogeochemical cycling of nutrients, carbon storage, soil aggregation, and transformation of toxic compounds in the source soil. Currently, much of the information on microbial transport in dust has been collected at continental scales, with no comprehensive review regarding the microbial communities, particularly those associated with agricultural systems, redistributed by wind erosion processes at smaller scales including regional or field scales. Agricultural systems can contribute significantly to atmospheric dust loading and loss or redistribution of soil microorganisms are impacted in three interactive ways: (1) differential loss of certain microbial taxa depending on particle size and wind conditions, (2) through the destabilization of soil aggregates and reduction of available surfaces, and (3) through the reduction of organic matter and substrates for the remaining community. The purpose of this review is to provide an overview of dust sampling technologies, methods for microbial extraction from dust, and how abiotic, environmental, and management factors influence the dust microbiome within and among agroecosystems. The review also offers a perspective on important potential future research avenues with a focus on agroecosystems and the inclusion of the fungal component.

Mdm2, an E3 ubiquitin ligase, negatively regulates the tumour suppressor p53. In this study we utilized a conditional Mdm2 allele, Mdm2(FM) , and a CAG-CreER tamoxifen-inducible recombination system to examine the effects of global Mdm2 loss in adult mice. Two different tamoxifen injection regimens caused 100% lethality of Mdm2(FM) (/-) ;CAG-CreER mice; both radio-sensitive and radio-insensitive tissues were impaired. Strikingly, a large number of radio-insensitive tissues, including the kidney, liver, heart, retina and hippocampus, exhibited various pathological defects. Similar tamoxifen injections in older (16-18 month-old) Mdm2(FM) (/-) ;CAG-CreER mice yielded abnormalities only in the kidney. In addition, transcriptional activation of Cdkn1a (p21), Bbc3 (Puma) and multiple senescence markers in young (2-4 month-old) mice following loss of Mdm2 was dampened in older mice. All phenotypes were p53-dependent, as Mdm2(FM) (/-) ;Trp53(-/-) ;CAG-CreER mice subjected to the same tamoxifen regimens were normal. Our findings implicate numerous possible toxicities in many normal tissues upon use of cancer therapies that aim to inhibit Mdm2 in tumours with wild-type p53.

Purpose C4, a cobalt dichloride-N-acetyl cysteine complex, is being developed as a positive-signal magnetic resonance imaging (MRI) marker to localize implanted radioactive seeds in prostate brachytherapy. We evaluated the toxicity and biodistribution of C4 in rats with the goal of simulating the systemic effects of potential leakage from C4 MRI markers within the prostate. Methods and Materials 9-μL doses (equivalent to leakage from 120 markers in a human) of control solution (0.9% sodium chloride), 1% (proposed for clinical use), and 10% C4 solution were injected into the prostates of male Sprague-Dawley rats via laparotomy. Organ toxicity and cobalt disposition in plasma, tissues, feces, and urine were evaluated. Results No C4-related morbidity or mortality was observed in the biodistribution arm (60 rats). Biodistribution was measurable after 10% C4 injection: cobalt was cleared rapidly from periprostatic tissue; mean concentrations in prostate were 163 μg/g and 268 μg/g at 5 and 30 minutes but were undetectable by 60 minutes. Expected dual renal-hepatic elimination was observed, with percentages of injected dose recovered in tissues of 39.0 ± 5.6% (liver), >11.8 ± 6.5% (prostate), and >5.3 ± 0.9% (kidney), with low plasma concentrations detected up to 1 hour (1.40 μg/mL at 5-60 minutes). Excretion in urine was 13.1 ± 4.6%, with 3.1 ± 0.54% recovered in feces by 24 hours. In the toxicity arm, 3 animals died in the control group and 1 each in the 1% and 10% groups from surgical or anesthesia-related complications; all others survived to scheduled termination at 14 days. No C4-related adverse clinical signs or organ toxicity were observed. Conclusion C4-related toxicity was not observed at exposures at least 10-fold the exposure proposed for use in humans. These data demonstrating lack of systemic toxicity with dual routes of elimination in the event of in situ rupture suggest that C4 warrants further investigation as an MRI marker for prostate brachytherapy.

Objective: The purpose of this study was to reduce the time to tumor onset in a diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) swine model via partial liver embolization (PLE) and to characterize the model for use in translational research. Methods: Eight Yucatan miniature pigs were injected intraperitoneally with either saline (n = 2) or DEN (n = 6) solution weekly for 12 weeks. Three of the DEN-treated pigs underwent PLE. The animals underwent periodic radiological evaluation, liver biopsy, and blood sampling, and full necropsy was performed at study termination (∼29 months). Results: All DEN-treated pigs developed hepatic adenoma and HCC. PLE accelerated the time to adenoma development but not to HCC development. Biomarker analysis results showed that IGF1 levels decreased in all DEN-treated pigs as functional liver capacity decreased with progression of HCC. VEGF and IL-6 levels were positively correlated with disease progression. Immunohistochemical probing of HCC tissues demonstrated the expression of several important survival-promoting proteins. Conclusion: To our knowledge, we are the first to demonstrate an accelerated development of hepatic neoplasia in Yucatan miniature pigs. Our HCC swine model closely mimics the human condition (i.e., progressive disease stages and expression of relevant molecular markers) and is a viable translational model.

Photothermal ablation (PTA) is an emerging technique that uses near-infrared (NIR) laser light-generated heat to destroy tumor cells. However, complete eradication of tumor cells with PTA is difficult because of uneven heat distribution in the treatment volume. We hypothesized that combining PTA with chemotherapy using a single multifunctional nanoconstruct that mediates simultaneous photothermal cell killing and drug release (photothermal-chemotherapy) would result in enhanced antitumor activity and reduced toxicity compared to chemotherapy alone. Doxorubicin (DOX) was loaded to hollow gold nanospheres (HAuNS) coated with polyethylene glycol (PEG). The pharmacokinetics and biodistribution of both DOX and HAuNS in the resulting nanoconstruct, DOX@PEG-HAuNS having different DOX:PEG:HAuNS ratios, were evaluated using dual isotope labeling techniques. The antitumor activity of DOX@PEG-HAuNS with DOX:PEG:HAuNS weight ratio of 1:3:1 (NP3) in combination with NIR laser was studied in vitro and in vivo using human MDA-MB-231 breast cancer and A2780 ovarian cancer cells. In vitro, NP3 mediated PTA of both cancer cells and DOX release upon NIR laser treatment. In vivo, NP3 showed slower clearance in blood and greater accumulation in tumors than free DOX. NP3-plus-NIR laser demonstrated greater antitumor activity than free DOX, NP3, or liposomal DOX. Moreover, NP3 displayed significantly decreased systemic toxicity compared to free DOX or liposomal DOX. Enhanced antitumor effect with NP3-plus-laser can be attributed to both the cytotoxic effect of DOX released from NP3 and the photothermal effect mediated by HAuNS. Slow release of DOX from NP3 in normal tissues contributed to reduced systemic toxicity. Photothermal-chemotherapy exemplified by a single-agent nanoconstruct NP3 is a promising approach to anticancer therapy.

The pharmacokinetic profile after hepatic arterial embolization with superabsorbent microspheres (QuadraSpheres) loaded with doxorubicin was studied. Rabbits with hepatic VX2 tumors were treated with intra-arterial administration of QuadraSpheres loaded with doxorubicin, or transarterial chemoembolization (TACE) using doxorubicin, Lipiodol and Embospheres, or hepatic arterial infusion (HAI) of doxorubicin. Tumor specimens were evaluated by fluorescence microscopy, and plasma and tumor concentrations of doxorubicin were measured. The peak plasma concentration of doxorubicin was lower in the QuadraSphere group (309.9 ng/ml) than in the HAI (673.4 ng/ml) or TACE (360.5 ng/ml) groups, suggesting higher tumor retention in the QuadraSphere group. Intratumoral doxorubicin levels declined to negligible levels at 1 and 3 days after treatment, respectively, in the HAI and TACE groups. In the QuadraSphere groups, intratumoral doxorubicin level declined after day 1, but was still detectable at 14 days after treatment and was higher than that in the other groups at 1, 3, and 7 days. Intratumoral doxorubicin fluorescence was detected at all time points in the QuadraSphere group, but only at 1 day after treatment in the TACE group. Hepatic arterial administration of doxorubicin-loaded QuadraSpheres enables the sustained release of doxorubicin to hepatic tumors.

Connective tissue growth factor (CTGF) belongs to the CCN family of proteins, which share a similar modular structure (1–3). Depending on the cell type, CTGF has key biologic functions ranging from regulation of cell growth to tissue remodeling (4). Expression of CTGF is observed in the mesenchyme during embryonic development, and studies involving disruption of the CTGF gene indicate that it is required for the coordination of chondrogenesis (5) and angiogenesis during development of the skeleton, and also lung development (4). There is little or no detectable CTGF in normal adult skin tissue; however, during normal wound healing, CTGF expression is activated at the wound site and then switched off once the repair process is complete (6). CTGF levels are increased in fibrotic tissue, and its levels have been shown to correlate with disease severity, e.g., in scleroderma (7). The precise mechanism by which CTGF activates cellular changes and the triggers that elicit CTGF expression in fibrotic lesions are poorly understood. Recently, activation of transforming growth factor β (TGFβ) signaling has been implicated as playing a major role in fibrosis (8). To directly assess the role of sustained TGFβ signaling, we have previously generated mice that specifically express genetically modified TGFβ receptors in fibroblasts, thus modulating TGFβ signaling (9,10). For example, expression of constitutively active TGFβ receptor I in fibroblasts postnatally recapitulated major clinical, biochemical, and histologic features of the fibrotic pathology in the skin and small blood vessels. Transgenic mouse fibroblasts displayed high levels of CTGF expression, suggesting that both TGFβ and CTGF may have an important role in a sustained chronic fibrotic outcome (9). A similar conclusion was reached from the analysis of different human fibroproliferative diseases. Interestingly, high levels of CTGF expression have been detected within fibrotic lesions in scleroderma patients even in the absence of elevated TGFβ levels (7). The purpose of this study was to investigate whether CTGF alone causes fibrosis in intact animals and whether the effects of CTGF are mediated through activation of TGFβ signaling or through other signaling pathways. For this purpose, we generated mice in which overexpression of CTGF was targeted to fibroblasts. We report that these mice spontaneously developed a multiorgan fibrotic phenotype affecting skin, lung, and kidney and including vascular remodeling of small blood vessels. The resulting phenotype, at least in the skin and lung, had features of tissue fibrosis similar to those observed in scleroderma. Molecular and cell biology studies of isolated primary mesenchymal connective tissue cells revealed that forced expression of CTGF in fibro-blasts did not involve the canonical TGFβ pathway, but promoted the activation of several other signaling pathways and downstream transcriptional programs, resulting in disruption of connective tissue architecture that is replaced by increased extracellular matrix (ECM). These results suggest that CTGF and its downstream pathways could be targets for antifibrotic therapy.

Improving wind erosion prediction and control requires correct estimates of the suspended dust (ss) and saltation (sn) components from field erosion events. The objectives of this study were to (a) develop an improved methodology (HPS) to estimate the fractions of sn and ss in the sediment discharge, and (b) compare HPS predictions to those of LM (Leys and McTainsh, 1996) and CN (Chepil and Woodruff, 1978; Nickling, 1978) that included or FS (Fryrear and Saleh, 1993) that excluded size measurements of the trapped sediment. Sediment flux profiles from nine field studies in four states were used in the data analyses. The log form of the ss flux profile developed in the LM method for dispersed particles had poor data fit to profiles of aggregated particles. The FS, HPS, and CN methods predicted significantly different ratios of ss discharge to total discharge in the order FS < HPS < CN. The widely-used power form of the ss flux profile in the CN method provided a good data fit above 0.1 m, but over estimated ss flux near the surface. The FS method over estimated sn and under estimated ss components. The HPS method obtained good fits to the data, even when profile gradients were large. Thus, using the sieved sediment catch from passive sediment catchers coupled with HPS analysis methodology offered significant improvements in the accuracy of estimates of sn and ss discharge. On short fields, the fraction of ss discharge was related to soil texture. Published by Elsevier B.V.

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences.

Lung cancers develop via multiple genetic and epigenetic changes, including inactivation of tumor suppressor genes. We previously cloned human G protein-coupled receptor family C type 5A (GPRC5A), whose expression is suppressed in some human lung carcinoma cells, and its mouse homolog Gprc5a.We generated Gprc5a knockout mice by homologous recombination and studied their phenotype by macroscopic observation and microscopic histologic analysis of embryos and lungs of 1- to 2-year-old mice. GPRC5A mRNA expression was analyzed by reverse transcription-polymerase chain reaction in surgical specimens of 18 human lung tumors and adjacent normal tissues and by analyzing previously published data from 186 lung tumor tissues of a variety of histologic types and 17 normal lung samples. Human embryonic kidney, human non-small-cell lung cancer, and mouse lung adenocarcinoma cells were transfected with a GPRC5A expression vector or a control vector, and colony formation in semisolid medium was assayed. Statistical tests were two-sided.Homozygous knockout mice developed many more lung tumors at 1-2 years of age (incidence: 76% adenomas and 17% adenocarcinomas) than heterozygous (11% adenomas) or wild-type (10% adenomas) mice. Human GPRC5A mRNA levels were lower in most (11 of 18 [61%]) human lung tumors than in adjacent normal tissues. The mean GPRC5A mRNA level in adenocarcinoma (n = 139), squamous cell carcinoma (n = 21), small-cell lung cancer (n = 6), and carcinoid (n = 20) tissues was 46.2% (P = .014), 7.5% (P.001), 5.3% (P.001), and 1.8% (P.001), respectively, that in normal lung tissues (n = 17) GPRC5A transfection suppressed colony formation in semisolid medium of immortalized human embryonic kidney, human non-small-cell lung cancer, and mouse lung adenocarcinoma cells by 91%, 91%, and 68%, respectively, compared with vector controls (all P.001).Gprc5a functions as a tumor suppressor in mouse lung, and human GPRC5A may share this property. The Gprc5a-deficient mouse is a novel model to study lung carcinogenesis and chemoprevention.

In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor–bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy. [Cancer Res 2007;67(15):7212–22]

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports ofin vivoinactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.

Deposition of suspended dust near eroding source fields can have detrimental effects on vegetation, as well as on soil and water quality. This study was undertaken to quantify dust deposition within 200 m of a source field during wind erosion events. Erosion was measured with BSNE samplers on a small field of Amarillo fine sandy loam at field at Big Spring, TX. Suspension-sized dust discharge averaged 33 ± 5 per cent of the total sediment discharge and ranged from 18·0 to 147·4 kg m−1 during eight selected storm events. Within 200 m of the source field boundary, dust collected in deposition samplers placed above a vegetated surface averaged 34 per cent of initial dust discharge. Predicted deposition, according to a line source model, was 43 per cent. Actual deposition was likely near that predicted, because of lateral diffusion of the dust and some under-sampling by the disk samplers. Thus, the line source model seems useful in estimating both the pattern and quantity of deposition. About 30 per cent of the suspended dust was deposited within the initial 50 m of vegetated surface, but only about 12–15 per cent was deposited in the initial 10 m. Published in 2006 by John Wiley & Sons, Ltd.

Loss of Mdm2 or Mdm4 leads to embryo lethal phenotypes that are p53-dependent. To determine whether Mdm2 and Mdm4 inhibit p53 function redundantly in a more restricted cell type, conditional alleles were crossed to a neuronal specific Cre transgene to delete Mdm2 and Mdm4 in the CNS. Mice lacking Mdm2 in the CNS developed hydranencephaly at embryonic day 12.5 due to apoptosis, whereas Mdm4 deletion showed a proencephaly phenotype at embryonic day 17.5 because of cell cycle arrest and apoptosis. The deletion of both genes, strikingly, contributed to an even earlier and more severe CNS phenotype. Additionally, Mdm2 and Mdm4 had a gene dosage effect, because loss of three of the four Mdm alleles also showed a more accelerated CNS phenotype than deletion of either gene alone. All phenotypes were rescued by deletion of p53 . Thus, these in vivo data demonstrate the importance of Mdm4 independent of Mdm2 in inhibition of p53.

The use of portable chemistry analyzers is an attractive option for obtaining clinical pathology panels in mice, because these analyzers require only small volumes of whole blood. However, in studies with other animals, portable analyzers do not always agree with results obtained using standard laboratory equipment. The authors evaluated the use of the i-STAT handheld portable clinical analyzer compared to the use of standard nonportable laboratory instruments in mice. As shown with other species, the i-STAT results did not always agree with standard laboratory instruments; however, the i-STAT does show reliability for certain chemistry assays.

PURPOSE To compare vascular injuries induced by a nonrotational thrombectomy device equipped with an adjustable basket (the AKonya Eliminator [AKE] device) and the Arrow-Trerotola percutaneous thrombolytic device (PTD) in porcine external iliac arteries (EIAs). MATERIALS AND METHODS The EIAs of nine domestic pigs underwent simulated thrombectomy with the AKE after the diameter of the basket had been adjusted to the vessel's diameter and with the PTD after motor activation. Three animals were euthanized immediately after treatment (group 1, acute), three after 1 week (group 2, subchronic), and three after 6 weeks (group 3, chronic). Vessel diameters were measured angiographically at four anatomic locations at the three time points. A histologic grading system was established to quantify the degree of vascular injury and lumen compromise. Four other EIAs were treated with an “oversized” AKE basket and followed for 6 weeks. RESULTS Histologically, the acute lesions in the AKE-treated vessels were more superficial than those in the PTD-treated vessels. In group 2, two of three PTD-treated arteries occluded, and their subchronic injuries were more serious than those in the AKE-treated arteries. In group 3, all AKE-treated arteries remained patent, but one of the PTD-treated vessels occluded, and the lumen sizes of the PTDand AKE-treated arteries differed significantly. After 6 weeks, there was no significant difference between arteries treated with the PTD and those treated with the oversized AKE in terms of diameter or histologic grading. CONCLUSION The adjustable basket and hand-controlled operation of the AKE were significantly less injurious to the arterial wall than the constant-size PTD basket operated at 3,000 rpm. Damage produced by the oversized AKE basket was similar to that produced by the PTD.

Tobacco carcinogens induce Akt activation and lung carcinogenesis. We previously demonstrated that deguelin, a natural plant product, specifi cally inhibits the proliferation of premalignant and malignant human bronchial epithelial cells by blocking Akt activation. To evaluate the ability of deguelin to block tobacco carcinogeninduced lung tumorigenesis, we evaluated the in vivo effects of deguelin on Akt activation and lung tumorigenesis in transgenic mice in which Akt expression was induced by tamoxifen and in 4-(methylnitrosoamino)-1-(3pyridyl)-1-butanone (NNK)/benzo(a) pyrene (BaP)-treated A/J mice. Deguelin suppressed Akt activation in vivo, as measured by immunohistochemistry and immunoblotting, and statistically signifi cantly reduced NNK/BaP-induced lung tumor multiplicity, volume, and load in A/J mice, as monitored by microcomputed tomography image analysis, with no detectable toxicity. These results indicate that deguelin warrants consideration as a chemopreventive agent for earlystage lung carcinogenesis in a clinical lung cancer chemoprevention trial. [J Natl Cancer Inst 2005;97:1695 – 9]

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D3 (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4 μg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in ∼10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

Contains fulltext : 33066.pdf (Publisher’s version ) (Closed access) We used a memory-saccade task to test whether the location of a target, briefly presented before a whole-body rotation in roll, is stored in egocentric or in allocentric coordinates. To make this distinction, we exploited the fact that subjects, when tilted sideways in darkness, make systematic errors when indicating the direction of gravity (an allocentric task) even though they have a veridical percept of their self-orientation in space. We hypothesized that if spatial memory is coded allocentrically, these distortions affect the coding of remembered targets and their readout after a body rotation. Alternatively, if coding is egocentric, updating for body rotation becomes essential and errors in performance should be related to the amount of intervening rotation. Subjects (n = 6) were tested making saccades to remembered world-fixed targets after passive body tilts. Initial and final tilt angle ranged between -120 degrees CCW and 120 degrees CW. The results showed that subjects made large systematic directional errors in their saccades (up to 90 degrees ). These errors did not occur in the absence of intervening body rotation, ruling out a memory degradation effect. Regression analysis showed that the errors were closely related to the amount of subjective allocentric distortion at both the initial and final tilt angle, rather than to the amount of intervening rotation. We conclude that the brain uses an allocentric reference frame, possibly gravity-based, to code visuospatial memories during whole-body tilts. This supports the notion that the brain can define information in multiple frames of reference, depending on sensory inputs and task demands.

To determine the temporal histopathologic findings associated with selective arterial injection of a 1:1 ethiodized oil-ethanol mixture (EEM) in normal rabbit kidney followed by administration of pure ethanol into the main renal artery.In five rabbits, the EEM was injected sequentially into each segmental renal artery of the right kidney until capillary stasis occurred. Pure ethanol was then injected into the main renal artery to achieve complete arterial stasis. Before sacrifice, the left kidney in each animal was acutely (ie, with a short follow-up period) embolized by using the same technique. The 10 kidneys of the five rabbits were evaluated microscopically at 1 (n = 3), 1(1/2) (n = 1), and 3 hours (n = 1) and 1 (n = 1), 3 (n = 1), 5 (n = 1), 7 (n = 1), and 14 days (n = 1) after embolization.Injection of the EEM (mean volume, 0.46 mL +/- 0.14 [SD]) followed by ethanol alone (mean volume, 0.25 mL +/- 0.09) led to complete stasis in all kidneys. There was no recanalization in the chronically (ie, with a longer follow-up period) embolized kidneys. Microscopically, uniform distribution of the EEM was evident in all slices at all time points. From 1 to 3 hours, sloughing of endothelium, formation of thrombi, and deposition of eosinophilic material along the renal, interlobar, and arcuate arteries were observed, without evidence of parenchymal damage. Within 24 hours, complete coagulative necrosis of the entire kidney occurred as a result of an occluding thrombus in the main renal artery. Analysis at subsequent time points revealed liquefaction of necrotic tissue and replacement with granulation tissue.In the rabbit, selective renal arterial injection of EEM followed by administration of ethanol produces vascular endothelial damage initiating thrombosis that results in renal infarction and ablation within 24 hours.

Contains fulltext : 139811.pdf (Publisher’s version ) (Open Access) Neural oscillations have emerged as one of the major electrophysiological phenomena investigated in cognitive and systems neuroscience. These oscillations are typically studied with regard to their amplitude, phase, and/or phase coupling. Here we demonstrate the existence of another property that is intrinsic to neural oscillations but has hitherto remained largely unexplored in cognitive and systems neuroscience. This pertains to the notion that these oscillations show reliable diversity in their phase-relations between neighboring recording sites (phase-relation diversity). In contrast to most previous work, we demonstrate that this diversity is restricted neither to low-frequency oscillations nor to periods outside of sensory stimulation. On the basis of magnetoencephalographic (MEG) recordings in humans, we show that this diversity is prominent not only for ongoing alpha oscillations (8-12 Hz) but also for gamma oscillations (50-70 Hz) that are induced by sustained visual stimulation. We further show that this diversity provides a dimension within electrophysiological data that, provided a sufficiently high signal-to-noise ratio, does not covary with changes in amplitude. These observations place phase-relation diversity on the map as a prominent and general property of neural oscillations that, moreover, can be studied with noninvasive methods in healthy human volunteers. This opens important new avenues for investigating how neural oscillations contribute to the neural implementation of cognition and behavior. 8 p.

Objective Gold nanoparticles have attracted enormous interest as potential theranostic agents. However, little is known about the long-term elimination and systemic toxicity of gold nanoparticles in the literature. Hollow gold nanospheres (HAuNS) is a class of photothermal conducting agent that have shown promises in photoacoustic imaging, photothermal ablation therapy, and drug delivery. It’s very necessary to make clear the biosafety of HAuNS for its further application. Methods We investigated the cytotoxicity, complement activation, and platelet aggregation of polyethylene glycol (PEG)-coated HAuNS (PEG-HAuNS, average diameter of 63 nm) in vitro and their pharmacokinetics, biodistribution, organ elimination, hematology, clinical chemistry, acute toxicity, and chronic toxicity in mice. Results PEG-HAuNS did not induce detectable activation of the complement system and did not induce detectable platelet aggregation. The blood half-life of PEG-HAuNS in mice was 8.19 ± 1.4 hr. The single effective dose of PEG-HAuNS in photothermal ablation therapy was determined to be 12.5 mg/kg. PEG-HAuNS caused no adverse effects after 10 daily intravenous injections over a 2-week period at a dose of 12.5 mg/kg per injection (accumulated dose: 125 mg/kg). Quantitative analysis of the muscle, liver, spleen, and kidney revealed that the levels of Au decreased 45.2%, 28.6%, 41.7%, and 40.8%, respectively, from day 14 to day 90 after the first intravenous injection, indicating that PEG-HAuNS was slowly cleared from these organs in mice. Conclusion Our data support the use of PEG-HAuNS as a promising photothermal conducting agent.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17 beta-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.

Purpose Chromosomal aberrations and the inflammatory phenotype have been identified as predictive factors for survival of uveal melanoma (UM). The mechanism by which these factors are linked remains obscure. We took the human leukocyte antigen (HLA), located on chromosome 6p as marker for inflammation and studied whether aberrations of chromosome 6p influenced the HLA expression in UM. Methods SNP-array copy number analysis and gene expression profiling were performed on 28 uveal melanomas of patients who underwent enucleation between 1999 and 2004 at the Leiden University Medical Center, Leiden, in The Netherlands. UM protein expression of HLA-A, -B and –DR was measured with immunohistochemistry. The status of chromosome 3, 6p, and gene expression of HLA and HLA regulators were analyzed, as well as protein expression of HLA. Results Gain of 6p was present in 8 cases (29%) and not related with survival. An increased gene expression was seen in the group who died due to metastatic UM for HLA-A and –B, and B2M. Gene expression correlated with protein expression for HLA-A, HLA-B, but not with HLA-DR. Tumors did not differ in level of HLA protein expression, HLA gene expression, and HLA regulator gene expression with regard to chromosome 6p status. Conclusion High HLA gene- and protein expression in UM are not influenced by gain of 6p. Yet for the first time we demonstrated that in UM an increased expression of HLA class I genes correlated with the elevated protein expression of HLA class I.

Purpose The presence of epithelial cells is a bad prognostic factor in uveal melanoma (UM) and epithelial tumors are characterized by high expression of E-cadherin. However, CDH1, the gene encoding for E-cadherin, is located at chromosome 16q which is lost in UM with a bad prognosis. In order to solve this paradox we studied the underlying epigenetic mechanism of the spindle to epithelial transition and the accompanying E-cadherin expression. Methods UM cell lines were in vitro treated with methylation and deacetylation inhibitors, or were cultured under hypoxic conditions to mimic in vivo UM conditions to induce epigenetic reprogramming. RNA and DNA was isolated for gene expression, DNA methylation, and chromosome structure analysis, while cell lysates were used for E-cadherin analysis. Results Treatment with methylation and deacetylation inhibitors resulted in a phenotypic change from a spindle to more epithelial cell type. Furthermore, inhibition of methylation and hypoxia treatment both upregulated E-cadherin expression in vitro and induced euchromatin formation at 16q22, where CDH1 is located. Conclusion By revealing epigenetic regulation of CDH1 in UM we resolve a step in the dynamic process of spindle to epitheloid celltype switch in UM. Since this switch could be induced by hypoxia we establish a link between environmental stress and progression. With these data we provide evidence for epigenetic regulation of UM progression as well as an option for future treatment.

We have combined several endpoints into a single 5-day in vivo screening procedure to identify estrogenic compounds and dopaminergic modulators, both of which play important roles in enhancing mammary tumorigenesis in rodents. The endpoints evaluated as markers of estrogenicity included increases in uterine fluid and vaginal cornification incidence, serum prolactin levels, uterine weight, uterine epithelial cell height, uterine stromal cell proliferation, and uterine progesterone receptor (PR) number and decreases in uterine estrogen receptor (ER) number. The endpoints evaluated for changes in dopamine regulation included increases in prolactin and decreases in growth hormone levels. The estrogen agonists estradiol (E2) and estriol (E3), the mixed estrogen agonist/antagonist tamoxifen (TAM), the full antiestrogen ICI-182,780 (ICI), and the dopamine modulators haloperidol (HAL) and reserpine (RES) were tested using a three-time/day (8-hr intervals) intraperitoneal dosing regimen in sexually mature ovariectomized female Crl:CD BR rats. All compounds were evaluated over a range of concentrations. This in vivo battery was used to evaluate the effects of different classes of endocrine modulators on the selected endpoints. For example, the estrogen receptor agonists E2 and E3 display a unique profile based on changes in the uterotrophic endpoints (estrus conversion, uterine fluid imbibition, increases in uterine weight, and uterine endometrial cell proliferation) where full and partial agonists can be distinguished by the magnitude of these responses. Both the estrogen receptor antagonist ICI and the dopamine modulators HAL and RES lack these uterotrophic responses. Dopamine modulators can be distinguished from estrogen receptor agonists by the profile of increased prolactin levels with no uterotrophic changes. Estrogen receptor antagonists can be distinguished from agonists by comparing their effects on ER, PR, and uterotrophic responses. For instance, the full estrogen receptor antagonist ICI decreased ER (to almost 0) and PR levels, but has no uterotrophic effects, while TAM decreases ER (to almost 0) and increases PR with uterotrophic effects. The most useful endpoints for distinguishing estrogen agonists and dopamine modulators were uterine fluid imbibition, uterine weight, uterine stromal cell proliferation, and serum prolactin levels. In order to distinguish an estrogen agonist from an antagonist, other endpoints, such as receptor levels, are necessary. The advantage of an in vivo screen is that it utilizes a metabolically and physiologically defined system which is especially important with highly integrative systems such as the endocrine system. This battery can be used as a screening tool to identify potential endocrine modulators as well as to identify mode of action following adverse findings in toxicology studies. Last, additional endpoints may be added to identify other classes of endocrine modulators.

Contains fulltext : 103489.pdf (Publisher’s version ) (Open Access) Many aspects of brain processing are intimately linked to brain rhythms. Essentially all classical brain rhythms, i.e., delta, theta, alpha, beta, and sleep waves, are highly heritable. This renders brain rhythms an interesting intermediate phenotype for cognitive and behavioral traits. One brain rhythm that has been particularly strongly linked to cognition is the gamma rhythm: it is involved in attention, short- and long-term memory, and conscious awareness. It has been described in sensory and motor cortices, association and control structures, and the hippocampus. In contrast to most other brain rhythms, the gamma frequency highly depends on stimulus and task conditions, suggesting a low heritability. However, the heritability of gamma has not been assessed. Here, we show that visually induced gamma-band synchronization in humans is strongly genetically determined. Eighty twin subjects (20 monozygotic and 20 dizygotic twin pairs) viewed a moving sinusoidal grating while their brain activity was recorded using magnetoencephalography. The stimulus induced spectrally confined gamma-band activity in sensors over visual cortex in all subjects, with individual peak frequencies ranging from 45 to 85 Hz. Gamma-band peak frequencies were highly correlated across monozygotic twins (r = 0.88), but not across dizygotic twins (r = 0.32) or unrelated subjects (r = 0.02). This implies a heritability of the gamma-band frequency of 91%. This strong genetic determination suggests that gamma-related cognitive functions are under close genetic control.

Chronic Toxicity/Oncogenicity of Dimethylformamide in Rats and Mice Following Inhalation Exposure. Malley, L. A., Slone, T. W., Jr., Van Pelt, C., Elliott, G. S., Ross, P. E., Stadler, J. C., and Kennedy, G. L., Jr. (1994). Fundam. Appl. Toxicol. 23, 268-279. The potential chronic toxicity and oncogenicity of dimethylformamide (DMF) was evaluated by exposing male and female rats and mice to 0, 25, 100, or 400 ppm DMF for 6 hr/day, 5 days/week for 18 months (mice) or 2 years (rats). Clinical pathology was evaluated at 3, 6, 12, 18 and 24 (rats only) months. An interim euthanasia for rats occurred at 12 months and hepatic cell proliferation in rats and mice was examined at 2 weeks, 3 months, and 12 months. No compound-related effects on clinical observations or survival were observed. Body weights of rats exposed to 100 (males only) and 400 ppm were reduced. Conversely, body weights were increased in 400 ppm mice. No hematologic changes were observed in either species. Serum sorbitol dehydrogenase activity was increased in rats exposed to 100 or 400 ppm. There were no compound-related effects on the estrous cycle of rats or mice at any concentration. Compound-related morphological changes were observed only in the liver. In rats, exposure to 100 and 400 ppm produced increased relative liver weights, centrilobular hepatocellular hypertrophy, lipofuscin/hemosiderin accumulation in Kupffer cells, and centrilobular single cell necrosis (400 ppm only). In mice, increased liver weights (100 ppm males, 400 ppm both sexes), centrilobular hepatocellular hypertrophy, accumulation of lipofuscin/hemosiderin in Kupffer cells, and centrilobular single necrosis were observed in all exposure groups. These observations occurred in a dose-response fashion and were minimal at 25 ppm. No increase in hepatic cell proliferation was seen in mice or female rats. Slightly higher proliferation was seen in male rats exposed to 400 ppm at 2 weeks and 3 months but not at 12 months. Dimethylformamide was not oncogenic under these experimental conditions in either the rat or mouse.

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status. Cancer Res; 71(23); 7168–75. ©2011 AACR.

The IGF axis has been implicated in the risk of various cancers. We previously reported a potential role of tissue-derived IGF in lung tumor formation and progression. However, the role of IGF-binding protein (IGFBP)-3, a major IGFBP, on the activity of tissue-driven IGF in lung cancer development is largely unknown. Here, we show that IGF-I, but not IGF-II, protein levels in non-small-cell lung cancer (NSCLC) were significantly higher than those in normal and hyperplastic bronchial epithelium. We found that IGF-I and IGFBP-3 levels in NSCLC tissue specimens were significantly correlated with phosphorylated IGF-IR (pIGF-IR) expression. We investigated the impact of IGFBP-3 expression on the activity of tissue-driven IGF-I in lung cancer development using mice carrying lung-specific human IGF-I transgene (Tg), a germline-null mutation of IGFBP-3, or both. Compared with wild-type (BP3(+/+)) mice, mice carrying heterozygous (BP3(+/-)) or homozygous (BP3(-/-)) deletion of IGFBP-3 alleles exhibited decreases in circulating IGFBP-3 and IGF-I. Unexpectedly, IGF(Tg) mice with 50% of physiological IGFBP-3 (BP3(+/-); IGF(Tg)) showed higher levels of pIGF-IR/IR and a greater degree of spontaneous or tobacco carcinogen [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung tumor development and progression than did the IGF(Tg) mice with normal (BP3(+/+;) IGF(Tg)) or homozygous deletion of IGFBP-3 (BP3(-/-); IGF(Tg)). These data show that IGF-I is overexpressed in NSCLC, leading to activation of IGF-IR, and that IGFBP-3, depending on its expression level, either inhibits or potentiates IGF-I actions in lung carcinogenesis.

Cyclin E activates Cdk2, controls centrosome duplication, and regulates histone gene transcription. Cyclin E is deregulated in cancer and appears as low-molecular-weight (LMW) isoforms that correlate strongly with decreased survival in breast cancer patients. Transgenic mice overexpressing LMW-cyclin E have increased incidence of mammary tumors and distant metastasis when compared with mice that had full-length cyclin E. To specifically test the requirement for Cdk2 in LMW-cyclin E–mediated mammary tumorigenesis, we generated transgenic mice, which expressed LMW-cyclin E in a Cdk2-deficient background. We found that mammary gland development proceeds relatively normally in these animals, indicating that Cdk2 kinase activity is largely dispensable for this process. However, Cdk2-deficient mice were completely resistant to LMW-cyclin E–mediated mammary tumors. Cdk2 wild-type or heterozygous mice succumbed to mammary tumors with mean latencies of 16 and 19.5 months, respectively, but Cdk2 nullizygous littermates did not display tumors through 24 months. Similarly, continuous administration of two different Cdk inhibitors significantly delayed LMW-cyclin E–induced mammary tumor progression. Triple transgenic mice generated in a p53 heterozygous background also displayed no tumors. Finally, we found that Cdk2 silencing induced cell death in LMW-overexpressing breast cancer cell lines, but not in cell lines lacking LMW expression. Our findings establish a requirement for Cdk2 in LMW-cyclin E–mediated mammary tumorigenesis, arguing that human breast tumors overexpressing LMW-cyclin E are prime candidates for anti-Cdk2 therapy. Cancer Res; 71(9); 3377–86. ©2011 AACR.