Henrik Daa Schrøder

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

•  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects. •  The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein, and complex I–IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres). •  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content. •  mtDNA was found to be a poor biomarker of mitochondrial content. •  Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.

Changes in satellite cells in human skeletal muscle after a single bout of high intensity exercise

No studies to date have reported activation of satellite cells in vivo in human muscle after a single bout of high intensity exercise. In this investigation, eight individuals performed a single bout of high intensity exercise with one leg, the contralateral leg being the control. A significant increase in mononuclear cells staining for the neural cell adhesion molecule (N‐CAM) and fetal antigen 1 (FA1) were observed within the exercised human vastus lateralis muscle on days 4 and 8 post exercise. In addition, a significant increase in the concentration of the FA1 protein was determined in intramuscular dialysate samples taken from the vastus lateralis muscle of the exercising leg (day 0: 1.89 ± 0.82 ng ml−1; day 2: 1.68 ± 0.37 ng ml−1; day 4: 3.26 ± 1.29 ng ml−1, P < 0.05 versus basal; day 8: 4.68 ± 2.06 ng ml−1, P < 0.05 versus basal and control). No change was noted in the control leg. Despite this increase in N‐CAM‐ and FA1‐positive mononuclear cells, an increased expression of myogenin and the neonatal isoform of the myosin heavy chain (MHCn) was not observed. Interestingly, myofibre lesions resulting from extensive damage to the proteins within the myofibre, particularly desmin or dystrophin, were not observed, and hence did not appear to induce the expression of either N‐CAM or FA1. We therefore propose that satellite cells can be induced to re‐enter the cell growth cycle after a single bout of unaccustomed high intensity exercise. However, a single bout of exercise is not sufficient for the satellite cell to undergo terminal differentiation.

Teratoma Formation by Human Embryonic Stem Cells Is Site Dependent and Enhanced by the Presence of Matrigel

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.

Radiation-induced brachial plexopathy: Neurological follow-up in 161 recurrence-free breast cancer patients

PURPOSE The purpose was to assess the incidence and clinical manifestations of radiation-induced brachial plexopathy in breast cancer patients, treated according to the Danish Breast Cancer Cooperative Group protocols. METHODS AND MATERIALS One hundred and sixty-one recurrence-free breast cancer patients were examined for radiation-induced brachial plexopathy after a median follow-up period of 50 months (13-99 months). After total mastectomy and axillary node sampling, high-risk patients were randomized to adjuvant therapy. One hundred twenty-eight patients were treated with postoperative radiotherapy with 50 Gy in 25 daily fractions over 5 weeks. In addition, 82 of these patients received cytotoxic therapy (cyclophosphamide, methotrexate, and 5-fluorouracil) and 46 received tamoxifen. RESULTS Five percent and 9% of the patients receiving radiotherapy had disabling and mild radiation-induced brachial plexopathy, respectively. Radiation-induced brachial plexopathy was more frequent in patients receiving cytotoxic therapy (p = 0.04) and in younger patients (p = 0.04). The clinical manifestations were paraesthesia (100%), hypaesthesia (74%), weakness (58%), decreased muscle stretch reflexes (47%), and pain (47%). CONCLUSION The brachial plexus is more vulnerable to large fraction size. Fractions of 2 Gy or less are advisable. Cytotoxic therapy adds to the damaging effect of radiotherapy. Peripheral nerves in younger patients seems more vulnerable. Radiation-induced brachial plexopathy occurs mainly as diffuse damage to the brachial plexus.

A RNA antagonist of hypoxia-inducible factor-1α, EZN-2968, inhibits tumor cell growth

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the α-subunit of HIF-1 (HIF-1α), which occurs in response to hypoxia or activation of growth factor pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1α protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits the expression of HIF-1α mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC50, 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor cell growth. Additionally, down-regulation of HIF-1α protein by EZN-2968 led to reduction of its transcriptional targets and of human umbilical vein endothelial cell tube formation. In vivo, administration of EZN-2968 to normal mice led to specific, dose-dependent, and highly potent down-regulation of endogenous HIF-1α and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program. [Mol Cancer Ther 2008;7(11):3598–608]

Tumorigenic Heterogeneity in Cancer Stem Cells Evolved from Long-term Cultures of Telomerase-Immortalized Human Mesenchymal Stem Cells

Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had approximately 1.2 days doubling time with loss of contact inhibition. All retained 1,25-(OH)(2) vitamin D(3)-induced expression of osteoblastic markers: collagen type I, alkaline phosphatase, and osteocalcin. All shared INK4a/ARF gene locus deletion and epigenetic silencing of the DBCCR1 tumor suppressor gene. Despite in vitro commonality, only four of six clones shared the growth kinetics and 100% tumorigenicity of the parental population. In contrast, one clone consistently formed latent tumors and the other established tumors with only 30% penetrance. Changing the in vitro microenvironment to mimic in vivo growth aspects revealed concordant clonal heterogeneity. Latent tumor growth correlated with extracellular matrix entrapment of multicellular spheroids and high procollagen type III expression. Poor tumorigenicity correlated with in vitro serum dependence and high p27(Kip1) expression. Aggressive tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer.

Risk of relapse in childhood acute lymphoblastic leukemia is related to RBC methotrexate and mercaptopurine metabolites during maintenance chemotherapy. Nordic Society for Pediatric Hematology and Oncology.

PURPOSE During maintenance chemotherapy for childhood acute lymphoblastic leukemia (ALL), the cytotoxic metabolites of methotrexate (MTX polyglutamates) and mercaptopurine (6MP) (thioguanine nucleotides [6TGN]) accumulate intracellularly, including in erythrocytes (E-MTX and E-6TGN) with large interindividual variations. In the present Nordic Society for Pediatric Hematology and Oncology (NOPHO) study, the relation of E-MTX and E-6TGN to relapse risk was explored. PATIENTS AND METHODS Two hundred ninety-seven patients with non-B-cell ALL, aged 1 to 14 years, on oral MTX and 6MP had E-MTX and E-6TGN levels measured three to 35 (median, eight) and three to 75 (median, nine) times, respectively. For each patient, a mean of all E-MTX (mE-MTX) and E-6TGN (mE-6TGN) measurements was calculated, as well as the product of mE-MTX and mE-6TGN (mE-MTX-6TGN), since MTX and 6MP may have synergistic action. RESULTS For patients in remission, the median mE-MTX and mE-6TGN values were 4.7 nmol/mmol hemoglobin (Hgb) (range, 0.4 to 10.3) and 173 nmol/mmol Hgb (range, 58 to 846). With a median follow-up duration of 66 months for patients in remission, 64 patients relapsed. Cox regression analysis identified mE-MTX-6TGN and sex to be the most significant parameters to predict relapse (global P = .001). Factors that predicted a better prognosis were high mE-MTX 6TGN and female sex. Patients who had a mE-MTX-6TGN less than the product of the median mE-MTX and median mE-6TGN (813 [nmol/mmol Hgb]2) had a significantly poorer event-free survival (EFS) than did patients with higher values (5-year probability of EFS [pEFS5y], 0.70 v 0.86; P = .001). CONCLUSION The pharmacokinetics of MTX and 6MP may have significant influence on the risk of relapse. The value of dose adjustments by E-MTX and E-6TGN remains to be determined.

A cellular model system of differentiated human myotubes

The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc‐cultures. In order to identify the differentiation conditions which give a good survival of myotubes and a high grade of differentiation, Sc‐cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK‐activities in cultures differentiating in FCS‐supplemented media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between days 4–10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8‐myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with clear cross striation. We recommend the use of differentiated Sc‐cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation‐related changes will take place in the cells during this period of time.

Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript.

Because previous PCR‐based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real‐time quantitative PCR (RQ‐PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL‐AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ‐PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL‐AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and β2‐microglobulin (β2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed β2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day‐to‐day variation. In 12 samples from t(12;21)‐positive patients at diagnosis, the levels of the TEL‐AML1 fusion transcripts were found to vary up to 14‐fold after normalization to β2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL‐AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355–365, 1999.

Fiber types in the striated urethral and anal sphincters

SummarySeven normal human striated urethral and anal sphincters obtained by autopsy were examined using histochemical techniques. In both the urethral sphincter and the subcutaneous (s.c.) and superficial part of the anal sphincter a characteristic pattern with two populations of muscle fibers, abundant connective tissue, and numerous intramuscular nerves are seen. No spindles are observed. The muscle fibers, particularly the predominant type 1 fibers are very small (about 15 μm in diameter). The fiber characteristics of the sphincters indicate that these muscles have a capacity to produce sustained contractions and to react in stress conditions with fast increase in tension.