Value of work for employees with a chronic disease.

Background: Most people with a chronic disease value participation in work. Knowledge is limited, however, as to what extent employees with a chronic disease value participating in work, and the main reasons for this. Limited research is available on which specific factors contribute to the perceived value of work. Aims: To evaluate main reasons for, and the extent to which employees with a chronic disease value participation in work, and factors which motivate or demotivate employees in work. Methods: A survey of members of three large patient federations was performed. Respondents had a chronic disease and were of working age. The extent and reasons for valuing work were analysed using descriptive statistics; (de)motivating aspects were qualitatively analysed using specific software. Results: The 1683 respondents valued work with an average of 8 on a scale from 1 to 10 (1: 'work is not at all important to me' and 10: 'work is extremely important to me'). Most frequent reported reasons for valuing work were the provision of income, social contact and the ability to contribute to society. Motivational aspects for work were being financially independent, having positive social contact with colleagues or clients and having the ability to contribute to society. In contrast, negative social contact, performing useless work and having little autonomy demotivated people. Conclusions: Employed people with a chronic disease generally value work, mainly because it makes them financially independent, provides social contact and enables them to contribute to society.

ADAM10 and ADAM17 have opposite roles during sprouting angiogenesis.

During angiogenesis, endothelial tip cells start sprouting and express delta-like 4 (DLL4) downstream of vascular endothelial growth factor (VEGF). DLL4 subsequently activates Notch in the adjacent stalk cells suppressing sprouting. VEGF also activates A disintegrin and metalloproteases (ADAMs) that induce Notch ectodomain shedding. Although two major ADAMs, i.e. ADAM10 and ADAM17, have been implicated in Notch-signalling activation, their apparent different roles in angiogenesis have not been fully understood yet. The objective of this study was to determine the roles of ADAM10 and ADAM17 activity in angiogenesis. In mouse retinas, ADAM10 or γ-secretase inhibition induced vascular sprouting and density in vivo, whereas attenuation of both ADAM10 and ADAM17 activity produced the opposite phenotype. Retinal blood vessel analysis in ADAM17 hypomorphic mice confirmed the requirement for ADAM17 activity in angiogenesis. However, ADAM17 inhibition did not phenocopy blood vessel increase by Notch blockage. These observations suggest that ADAM17 regulates other fundamental players during angiogenesis besides Notch, which were not affected by ADAM10. By means of an angiogenesis proteome assay, we found that ADAM17 inhibition induced the expression of a naturally occurring inhibitor of angiogenesis Thrombospondin 1 (TSP1), whereas ADAM10 inhibition did not. Accordingly, ADAM17 overexpression downregulated TSP1 expression, and the TSP1 inhibitor LSKL rescued angiogenesis in the tube formation assay downstream of VEGF in the presence of ADAM17 inhibition. Finally, genetic and pharmacological ADAM17 blockade resulted in increased TSP1 expression in mouse retina. Altogether, our results show that ADAM10 and ADAM17 have opposite effects on sprouting angiogenesis that may be unrelated to Notch signalling and involves differentially expressed anti-angiogenic proteins such as TSP1.

Methylation is less abundant in BRCA1-associated compared with sporadic breast cancer.

BACKGROUND: Promoter methylation is a common epigenetic mechanism to silence tumor suppressor genes during breast cancer development. We investigated whether BRCA1-associated breast tumors show cancer-predictive methylation patterns similar to those found in sporadic tumors. PATIENTS AND METHODS: Quantitative multiplex methylation-specific PCR of 11 genes involved in breast carcinogenesis (RARB, RASSF1, TWIST1, CCND2, ESR1, SCGB3A1, BRCA1, BRCA2, CDKN2A, APC, CDH1) was carried out on 32 BRCA1-associated and 46 sporadic breast carcinomas and on normal breast tissue from seven BRCA1 mutation carriers and 13 non-carriers. RESULTS: The extent of cumulative methylation increased with age (P < 0.001). The median cumulative methylation index (CMI) of all studied genes was significantly higher in tumors (89) than in normal tissue (13, P < 0.001). The median CMI was significantly lower in BRCA1-associated (59) than in sporadic breast tumors (122, P = 0.001), in estrogen receptor (ER)-negative tumors (73) than in ER-positive tumors (122, P = 0.005) and in lymph node-negative (77) compared with lymph node-positive tumors (137, P = 0.007). In subgroup analysis, the effect of a BRCA1 germline mutation on methylation proved to be independent of ER status, lymph node status and age. CONCLUSIONS: These data indicate that BRCA1-associated breast cancers show less promoter methylation compared with sporadic breast carcinomas indicating a difference in disease etiology.

[Guideline 'The chronically ill and work']. / Richtlijn 'Chronisch zieken en werk'.

- The guideline 'The chronically ill and work' gives insight into disease-overarching factors and interventions that can promote or impede the participation in the work process of workers and those looking for work who have a chronic condition. - In particular, the guideline focuses on the role taken on by workers or those looking for work themselves during the process of keeping or resuming work. - The guideline gives recommendations for the daily practice of healthcare providers which are based on knowledge from disease-specific guidelines, the international literature and the experiences of healthcare providers, and workers and those looking for work with a chronic condition.

[Epigenetic processes in malignant transformation: the role of DNA methylation in cancer development]. / Epigenetische processen in de maligne ontaarding: de rol van DNA-methylering in het ontstaan van kanker.

Defects in DNA that activate oncogenes or inactivate tumour-suppressor genes are regarded as a crucial step in tumour development. Understanding the processes that modulate gene activity, the so-called epigenetic processes, is gaining importance in the search for factors responsible for uncontrolled cell growth. Cell proliferation is determined by epigenetic and genetic processes. Abnormal patterns of methylation and other epigenetic processes, such as acetylation, nucleosome formation and compact chromatin structure, can suppress transcription and inactivate tumour-suppressor genes. Methylation status is a promising biomarker for malignancy because the process is not patient-specific, it occurs at an early stage oftumour development and may precede morphological changes.

Developmental defects and tumor predisposition in Rb mutant mice.

Targeted gene disruption in the mouse germline permits the introduction of gene mutations similar to those found in inherited human diseases. New advances in gene targeting that enable cell type specific gene disruption in mice further increases the utility of mouse models to study genetic defects as found in cancer. Here we review the phenotypes observed in mice carrying germline mutated copies of the retinoblastoma tumor suppressor gene. We will illustrate how methods that permit tissue-specific Rb inactivation in mice provide new and more versatile tools to gain insight into the etiology of sporadic cancer.

Flp-mediated tissue-specific inactivation of the retinoblastoma tumor suppressor gene in the mouse.

The yeast-derived Flp-frt site-specific DNA recombination system was used to achieve pituitary-specific inactivation of the retinoblastoma (Rb) tumor suppressor gene. Whereas mice carrying only frt sites in both alleles of Rb remain tumor free, tumorigenesis ensues when the Flp recombinase is expressed. The rate of tumorigenesis in these mice depends both on the expression level of the Flp recombinase and on the presence of frt sites in one or both Rb alleles. This permitted a more accurate definition of the consecutive steps in pituitary tumorigenesis. Our study illustrates the potential of this approach for studying sporadic cancer in a defined mouse model.

The presence of endometrial cells in cervical smears in relation to the day of the menstrual cycle and the method of contraception.

The presence of endometrial cells in cervical smears was studied in a large series of women participating in a population screening program for cervical cancer, in relation to different time periods of the menstrual cycle and to the method of contraception practiced. In the total group of women studied, endometrial cells were present in an average of 12% of the cervical smears. In women who were menstruating cyclically, the percentage of cervical smears containing endometrial cells was not age dependent. Only in women over 52 years was a lower number of endometrium-positive cervical smears found: in postmenopausal women, 0.6% of smears were found to contain endometrial cells. In menstruating women, the frequency of endometrial cells in cervical smears was highest during the menses. After day four, through the proliferative phase, the percentages of cervical smears containing endometrial cells markedly decreased. During the secretory phase, an average of 2% of the smears contained endometrial cells; in the premenstrual phase (after day 25), the percentages of endometrial cell-positive smears rose again. When related to the method of contraception practiced, significant differences in the percentages of cervical smears with endometrial cells appeared. In women using oral hormonal contraceptives, the average numbers of smears containing endometrial cells for the whole cycle as well as for each period of the cycle were significantly lower. This phenomenon might be due to endometrial atrophy on the basis of prolonged use of oral hormonal contraceptives. In women wearing an intrauterine device, at any moment the frequencies of smears with endometrial cells present were significantly higher than the values found in women using any other method of contraception or not using contraceptives. The evaluation of cells originating from the endometrium requires considerable experience. The identification of endometrial cells can be made with greater confidence when the cytologist is aware of the exact date of the menstrual cycle and of the impact on the presence of endometrial cells in cervical smears caused by different methods of contraception.

FER kinase promotes breast cancer metastasis by regulating α6- and ß1-integrin-dependent cell adhesion and anoikis resistance.

Metastatic breast cancer cannot be treated successfully. Currently, the targeted therapies for metastatic disease are limited to human epidermal growth factor receptor 2 and hormone receptor antagonists. Understanding the mechanisms of breast cancer growth and metastasis is therefore crucial for the development of new intervention strategies. Here, we show that FER kinase (FER) controls migration and metastasis of invasive human breast cancer cell lines by regulating α6- and ß1-integrin-dependent adhesion. Conversely, the overexpression of FER in non-metastatic breast cancer cells induces pro-invasive features. FER drives anoikis resistance, regulates tumour growth and is necessary for metastasis in a mouse model of human breast cancer. In human invasive breast cancer, high FER expression is an independent prognostic factor that correlates with high-grade basal/triple-negative tumours and worse overall survival, especially in lymph node-negative patients. These findings establish FER as a promising target for the prevention and inhibition of metastatic breast cancer.

Proteolytic cleavage of Notch: "HIT and RUN".

The Notch pathway is a highly conserved signaling pathway in multicellular eukaryotes essential in controlling spatial patterning, morphogenesis and homeostasis in embryonic and adult tissues. Notch proteins coordinate cell-cell communication through receptor-ligand interactions between adjacent cells. Notch signaling is frequently deregulated by oncogenic mutation or overexpression in many cancer types. Notch activity is controlled by three sequential cleavage steps leading to ectodomain shedding and transcriptional activation. Here we review the key regulatory steps in the activation of Notch, from receptor maturation to receptor activation (HIT) via a rate-limiting proteolytic cascade (RUN) in the context of species-specific differences.

Mammary development and breast cancer: the role of stem cells.

The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.

Hypoxia-inducible factor 1 alpha is essential for hypoxic p27 induction in endometrioid endometrial carcinoma.

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia. The cyclin-dependent kinase inhibitor p27(Kip1) is highly expressed in the normal endometrium but is lost during endometrial carcinogenesis. However, in high-grade cancers, p27 re-expression is observed. We analysed the role of HIF-1alpha in hypoxia-induced expression of p27 in vitro and in vivo in endometrial cancer. Paraffin-embedded specimens from endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for HIF-1alpha, p27, and Ki67. HEC1B, an endometrial carcinoma cell line, was cultured under normoxic or hypoxic conditions in the presence or absence of transiently expressed short hairpin RNAs targeting HIF-1alpha. Protein expression of p27 and HIF-1alpha was assessed by western blotting. Immunohistochemical staining revealed perinecrotic HIF-1alpha expression in 67% of the cases and p27 staining centrally in the tumour islands, mostly around necrosis, in 46% of the cases. In 50% of the tumours with perinecrotic HIF-1alpha expression, p27 and HIF-1alpha perinecrotic/central co-localization was observed. In these tumour sections, hypoxia-associated p27 expression showed less proliferation around necrosis. Analysis of cultured endometrial carcinoma cells demonstrated that p27 protein expression is induced by hypoxia. This induction was abrogated by transient knockdown of HIF-1alpha using RNAi. Furthermore, hypoxia induced cell cycle arrest in HEC1B cells. We conclude that, in endometrioid endometrial carcinoma, p27 re-expression by hypoxia is HIF-1alpha-dependent and leads to cell cycle arrest. This may contribute to the survival of cancer cells in hypoxic parts of the tumour.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ERT) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.

Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum.

Medulloblastomas are among the most common malignancies in childhood, and they are associated with substantial mortality and morbidity. The molecular pathogenesis as well as the ontogeny of these neoplasms is still poorly understood. We have generated a mouse model for medulloblastoma by Cre-LoxP-mediated inactivation of Rb and p53 tumor suppressor genes in the cerebellar external granular layer (EGL) cells. GFAP-Cre-mediated recombination was found both in astrocytes and in immature precursor cells of the EGL in the developing cerebellum. GFAP-Cre;Rb(LoxP/LoxP);p53(-/- or LoxP/LoxP) mice developed highly aggressive embryonal tumors of the cerebellum with typical features of medulloblastoma. These tumors were identified as early as 7 weeks of age on the outer surface of the molecular layer, corresponding to the location of the EGL cells during development. Our results demonstrate that loss of function of RB is essential for medulloblastoma development in the mouse and strongly support the hypothesis that medulloblastomas arise from multipotent precursor cells located in the EGL.

Coagulation factor XIII in pregnant smokers and non-smokers.

Human blood coagulation factor XIII is a transglutaminase zymogen. Two forms exist, an extracellular or plasma factor XIII and an intracellular form. Factor XIII occurs in platelets, blood, monocytes, megakaryocytes, the liver, the placenta, and the uterus. In obstetrics, factor XIII deficiency has been associated with fetal wastage. The interaction of smoking and the quantity of coagulation factor XIII during normal pregnancy was examined in 75 non-smoking and 118 smoking (> or = 20 cigarettes/day) women. A group of subjectively healthy, non-smoking, age-matched females served as a control group (n = 30). Smokers had a higher plasma concentration of factor XIII than non-smokers. Factor XIII declined during normal gestation. During the second half of gestation the plasma concentration of factor XIII was significantly higher in smokers than in non-smokers. In smokers the decline of factor XIII was less, possibly due to platelet activation and a relative polycythemia. The later decline of factor XIII in pregnant smokers remains unexplained. More extensive research with larger patient numbers is needed to address this matter.

Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR.

We describe here the production of complex libraries enriched in sequences from each human chromosome type, starting with only a few thousand sorter-purified chromosomes. In this procedure, DNA is extracted from the sorted chromosomes, digested to completion by using the frequently cutting restriction endonuclease Sau3A1, and ligated, on each end, to an adaptor oligonucleotide. These fragments are then amplified using PCR with a sequence homologous to the adaptor oligonucleotide as a primer. We have used this procedure to produce PCR libraries for each of the 24 human chromosomes. These libraries were characterized by gel electrophoresis and found to be composed of a continuum of sequences ranging in size from a few hundred to approximately 1,000 bp. The libraries, when used as probes for fluorescence in situ hybridization, stained the target chromosomes more or less continuously, even after PCR amplification for more than 200 cycles. These libraries are useful as hybridization probes to facilitate molecular cytogenetic studies and as sources of probes either for identification of polymorphic short tandemly repeated sequences or for development of sequence-tagged sites.

A mouse chromosome 11 library generated from sorted chromosomes using linker-adapter polymerase chain reaction.

We describe the generation of a mouse whole chromosome library using sequence-independent polymerase chain reaction (PCR) to amplify sequences contained in DNA extracted from flow sorted chromosomes. DNA in sorted chromosomes from a human x mouse hybrid cell line was digested with a frequent four-cutter restriction enzyme, Sau3AI, and the ends were ligated to an adapter oligonucleotide. The ligated DNA fragments were amplified using PCR primers homologous to the linker-adapter oligonucleotide. PCR-generated products were characterized by gel electrophoresis. The size of the amplified DNA ranged from 100 to more than 1,000 bp with a relatively high proportion of products at approximately 400 bp. Biotinylated PCR products used for FISH showed specific hybridization to murine metaphases and no hybridization to human lymphocyte and hamster metaphase cells. Banding analysis indicated that the probes were specific for mouse Chromosome 11. We anticipate that availability of painting probes for specific murine chromosomes will facilitate cytogenetic studies in the mouse.

Cre-mediated somatic site-specific recombination in mice.

Conditional mutant mice equipped with heterologous recombination systems (Cre/lox or Flp/frt) are promising for studying tissue-specific gene function and for designing better models of human diseases. The utility of these mice depends on the cell target specificity, on the efficiency and on the control over timing of gene (in)activation. We have explored the utility of adenoviral vectors and transgenic mice expressing Cre under the control of tissue-specific promoters to achieve Cre/lox-mediated somatic recombination of the LacZ reporter gene, using a newly generated flox LacZ mouse strain. When adeno Cre viruses were administered via different routes, recombination and expression of LacZ was detected in a wide range of tissues. Whereas in liverbeta-galactosidase activity was quickly lost by turnover of expressing cells, even though the recombined allele was retained,beta-galactosidase in other tissues persisted for many months. Our data indicate that the flox LacZ transgenic line can be utilized effectively to monitor the level and functionality of Cre protein produced upon infection with adeno Cre virus or upon crossbreeding with different Cre transgenic lines.