Regulation of MYCN expression in human neuroblastoma cells

Background  

Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN -amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (ΔMYCN) that was recently identified in fetal tissues. Both MYCNOS and ΔMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level.     

Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene

Polycomb group (PcG) genes contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis. We describe the expression of five PcG genes (BMI-1, RING1, HPC1, HPC2, and EZH2) innormal breast tissues, invasive breast carcinomas, and their precursors. Members of the HPC-HPH/PRC1 PcG complex, including BMI-1, RING1, HPC1, and HPC2, were detected in normal resting and cycling breast cells. The EED-EZH/PRC2 PcG complex protein EZH2 was only found in rare cycling cells, whereas normal resting breast cells were negative for EZH2. PcG gene expression patterns in ductal hyperplasia (DH), well-differentiated ductal carcinoma in situ (DCIS), and well-differentiated invasive carcinomas closely resembled the pattern in healthy cells. However, poorly differentiated DCIS and invasive carcinomas frequently expressed EZH2 in combination with HPC-HPH/PRC1 proteins. Most BMI-1/EZH2 double-positive cells in poorly differentiated DCIS were resting. Poorly differentiated invasive carcinoma displayed an enhanced rate of cell division within BMI-1/EZH2 double-positive cells. We propose that the enhanced expression of EZH2 in BMI-1(+) cells contributes to the loss of cell identity in poorly differentiated breast carcinomas, and that increased EZH2 expression precedes high frequencies of proliferation. These observations suggest that deregulated expression of EZH2 is associated with loss of differentiation and development of poorly differentiated breast cancer in humans.     

Mycosis fungoides and Sézary syndrome: Current challenges in assessment,management and prognostic markers

Mycosis fungoides and Sézary syndrome are the most common variants of the cutaneous T‐cell lymphomas. Assessment of a patient with a suspected diagnosis requires thorough history taking and physical examination, in combination with skin biopsy. In some cases flow cytometry, molecular studies and imaging are also required in order to diagnose and stage the disease. Staging is derived from the tumour‐node‐metastasis‐blood classification and is currently our best attempt to stratify prognosis and hence guide management in this complex disease. Many other clinical, biological and pathological factors may help to distinguish groups at risk and predict prognosis more accurately. Management remains heavily guided by staging, such that patients with early‐stage disease generally begin treatment with skin‐directed or local therapies and those with advanced‐stage disease have many treatment options, including chemotherapy, the use of biological agents, local and total body radiotherapy, as well as haematopoietic stem cell transplantation. Besides staging, many other patient‐related factors influence the treatment strategy, particularly where symptom relief is paramount. There are many challenges remaining in the study of Mycosis fungoides and Sézary syndrome and, given the rarity of the disease, concerted worldwide efforts are required to conduct efficient and effective research.     

Does a reduced glucose intake prevent hyperglycemia in children early after cardiac surgery? a randomized controlled crossover study

Introduction

Hyperglycemia in children after cardiac surgery can be treated with intensive insulin therapy, but hypoglycemia is a potential serious side effect. The aim of this study was to investigate the effects of reducing glucose intake below standard intakes to prevent hyperglycemia, on blood glucose concentrations, glucose kinetics and protein catabolism in children after cardiac surgery with cardiopulmonary bypass (CPB).

Methods

Subjects received a 4-hour low glucose (LG; 2.5 mg/kg per minute) and a 4-hour standard glucose (SG; 5.0 mg/kg per minute) infusion in a randomized blinded crossover setting. Simultaneously, an 8-hour stable isotope tracer protocol was conducted to determine glucose and leucine kinetics. Data are presented as mean ± SD or median (IQR); comparison was made by paired samples t test.

Results

Eleven subjects (age 5.1 (20.2) months) were studied 9.5 ± 1.9 hours post-cardiac surgery. Blood glucose concentrations were lower during LG than SG (LG 7.3 ± 0.7 vs. SG 9.3 ± 1.8 mmol/L; P < 0.01), although the glycemic target (4.0-6.0 mmol/L) was not achieved. No hypoglycemic events occurred. Endogenous glucose production was higher during LG than SG (LG 2.9 ± 0.8 vs. SG 1.5 ± 1.1 mg/kg per minute; P = 0.02), due to increased glycogenolysis (LG 1.0 ± 0.6 vs. SG 0.0 ± 1.0 mg/kg per minute; P < 0.05). Leucine balance, indicating protein balance, was negative but not affected by glucose intake (LG -54.8 ± 14.6 vs. SG -58.8 ± 16.7 μmol/kg per hour; P = 0.57).

Conclusions

Currently recommended glucose intakes aggravated hyperglycemia in children early after cardiac surgery with CPB. Reduced glucose intake decreased blood glucose concentrations without causing hypoglycemia or affecting protein catabolism, but increased glycogenolysis.

Trial registration

Dutch trial register NTR2079.     

Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony- forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.     

Induced luteolysis in the primate: rapid loss of luteinizing hormone receptors

The molecular mechanisms involved in luteolysis are still unclear in the primate. This study aimed to investigate the effect of induced luteolysis on the ovarian luteinizing hormone (LH) receptor and the steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the marmoset monkey. Luteolysis was induced in the mid-luteal phase either directly by systemic prostaglandin F2alpha (PGF2alpha), or indirectly by LH withdrawal using systemic gonadotrophin releasing hormone antagonist (GnRHant) treatment. The LH receptor was studied by isotopic mRNA in-situ hybridization and in-situ ligand binding and 3beta-HSD expression was studied using isotopic mRNA in-situ hybridization and immunohistochemistry. Induced luteolysis was associated with a reduction in the expression of LH receptor (P < 0.0001) and 3beta-HSD mRNA, closely followed by a reduction in the LH receptor (P < 0.05) and 3beta-HSD protein concentrations within 24 h. There were no differences in the findings whether luteolysis was induced with PGF2alpha or GnRHant. This study shows that disparate mechanisms to induce luteolysis in the primate result in an identical rapid loss of the LH receptor and 3beta-HSD. In conclusion, induced luteolysis leads to rapid loss of the steroidogenic pathway in luteal cells.      

General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions.

We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV types X [HPV X]) in cervical smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n = 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types.     

Dentomaxillofacial manifestations of Gaucher's disease: preliminary clinical and radiographic findings

Objectives

A wide variety of manifestations is presented in patients with Gaucher''s disease (GD), including bone, haematology and visceral disturbances. This study was conducted to ascertain the main maxillofacial abnormalities by means of clinical survey, panoramic and cone beam CT (CBCT); to compare the patient''s group with an age–sex matched control group; and to correlate clinical and radiological data.

Methods

Ten patients previously diagnosed with GD were submitted to clinical and radiological surveys (CBCT and panoramic radiographs). The examination consisted of anamnesis, extra- and intraoral examinations and analyses of each patient''s records. Imaging data were collected from the point of view of 3 observers, and the results compared with a healthy group (20 individuals) by means of statistical analysis (Fisher''s exact test).

Results

Gaucher patients had significantly more manifestations than otherwise healthy carriers. The most prevalent findings were enlarged marrow spaces, generalized osteopenia and effacement of jaw structures (mandibular canal, lamina dura and mental foramen). Here we describe a case in which thickening of the maxillary sinus mucosa was observed on CBCT rather than opacification of the sinus as seen on panoramic radiographs. Pathological fractures, root resorption and delay on tooth eruption were not observed.

Conclusions

A poor relationship could be observed between clinical and radiological data. Patients showed important bone manifestations, which require careful diagnostic and surgical planning whenever necessary. Although panoramic radiographs have shown significant differences, CBCT is more effective in pointing out differences between patients and a control group, thus showing it as an important tool for evaluation of Gaucher patients.