Jacqueline Lyautey

About the possible origin and mechanism of circulating DNA apoptosis and active DNA release.

BACKGROUND In addition to cell lysis, apoptosis has been advanced as the origin of circulating DNA on the basis of several observations. Plasma or serum DNA often presents a ladder pattern reminiscent of that displayed by apoptotic cells when subjected to electrophoresis. However, the phenomenon of active release of DNA from cells might also be expected to result in a ladder pattern on electrophoresis. Non-dividing cells, such as lymphocytes, frog auricles and cultured cell lines including HL-60, spontaneously release a nucleoprotein complex within a homeostatic system in which newly synthesized DNA is preferentially released. CONCLUSION In relation to DNA synthesis, the phenomenon of extracellular DNA in different culture conditions favors apoptosis or spontaneous active DNA release.

Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia

Summary. Oncogene mutations are frequently found in several tumour types and, among these, point mutations of the ras gene are particularly significant. A predominance of N‐ras mutations has been found in the bone marrow DNA of patients with myelodysplatic syndrome (MDS) or acute myelogenous leukaemia (AML). On the other hand, increased levels of plasma DNA have previously been observed in patients suffering from various malignant diseases. In the present work we have investigated, by polymerase chain reaction (PCR), point mutations of the N‐ras gene in the DNA of plasma, blood cells and bone marrow of 10 patients suffering from AML or MDS. The different ras mutations detected in five cases were always present in the plasma DNA while sometimes absent in the DNA of peripheral blood cells or bone marrow. This indicates that a bone marrow biopsy or aspiration does not necessarily contain all the malignant clones involved in the disease. Plasma could thus prove to be an easily accessible and useful material for detection and monitoring of myeloid disorders.

Isolation and characterization of DNA from the plasma of cancer patients.

UNLABELLED Ten out of 37 patients with advanced malignant diseases were found to have extractable amounts of DNA in their plasma whereas no DNA could be detected in 50 normal controls. After its purification from the original nucleoprotein complex, DNA plasma levels ranging from 0.15 to 12 micrograms/ml were measured, the lowest concentration detectable with our method being 0.1 microgram/ml. Knowing from recovery experiments performed with 32P-DNA that the loss of DNA during the extraction procedure is about 65%, the real concentration of DNA in the plasma corresponds to about 3 times the given figures. The purified DNA was shown to be double-stranded and composed of fractions ranging from 21 kb to less than 0.5 kb, as determined by agarose gel electrophoresis. All these fractions hybridized with a 32P-labelled human DNA probe indicating the human origin of the bulk of the circulating DNA. IN CONCLUSION the finding of extractable amounts of DNA in the plasma of 27% of the investigated cancer patients, and its absence from the controls, suggests some correlation with malignancy.

The origin and mechanism of circulating DNA.

Although it is evident that DNA circulates freely in blood plasma both in disease and in health, the source of this DNA remains enigmatic. It can be presumed that circulating DNA in healthy subjects derives from lymphocytes or other nucleated cells. Yet, it is not known why cancer patients have such large quantities of plasma DNA. There is no doubt that a proportion seems to originate from nucleated blood cells since wild-type DNA has been detected in the plasma of all cancer patients studied as well as in that of healthy controls. However, a substantial proportion of plasma DNA in cancer patients derives from tumor cells. This latter concept is supported by both quantitative and qualitative observations. Plasma DNA levels are not only greater in cancer patients than in normal subjects,1–5 but also correlate inversely with outcome and tend to fall with effective treatment.1,5 In addition, the ability to detect LOH in plasma DNA6–13 suggests that tumor-related DNA is the predominant subtype in at least some cancer patients. What is responsible for the presence of tumor DNA shed in the bloodstream? It could be due to lysis of circulating cancer cells or of micrometastases, to DNA leakage resulting from tumor necrosis or apoptosis, or to a new mechanism of active release.

K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer

BACKGROUND & AIMS Circulating DNA can be isolated from the plasma of healthy subjects and from patients with cancer. The aim of this study was to detect K-ras mutations in DNA extracted from the plasma of patients with colorectal cancer. METHODS Tumor and plasma DNA were extracted from 14 patients with colorectal cancer (stages A-D), and K-ras alterations were detected using a polymerase chain reaction assay that uses sequence-specific primers to amplify mutant DNA. These results were confirmed with another polymerase chain reaction assay that creates an enzyme restriction site in the absence of a K-ras mutation followed by direct sequencing and additional cloning techniques. RESULTS Seven patients (50%) had a codon 12 K-ras mutation within their primary tumor, and identical mutations were found in the plasma DNA of 6 patients (86%). Mutant DNA was not detected in the plasma specimens of 7 patients whose tumors tested negative for K-ras alterations or in healthy control subjects. Similar results were obtained using all three molecular biological techniques. CONCLUSIONS K-ras abnormalities can be detected in circulating DNA extracted from the plasma specimens of patients with colorectal cancer. If these results are confirmed in larger studies, genetic analysis of plasma DNA may have clinical applications in the future.

Bucky Ball Organizes Germ Plasm Assembly in Zebrafish

In many animals, gamete formation during embryogenesis is specified by maternal cytoplasmic determinants termed germ plasm. During oogenesis, germ plasm forms a distinct cellular structure such as pole plasm in Drosophila or the Balbiani body, an aggregate of organelles also found in mammals. However, in vertebrates, the key regulators of germ plasm assembly are largely unknown. Here, we show that, at the beginning of zebrafish oogenesis, the germ plasm defect in bucky ball (buc) mutants precedes the loss of polarity, indicating that Buc primarily controls Balbiani body formation. Moreover, we molecularly identify the buc gene, which is exclusively expressed in the ovary with a novel, dynamic mRNA localization pattern first detectable within the Balbiani body. We find that a Buc-GFP fusion localizes to the Balbiani body during oogenesis and with the germ plasm during early embryogenesis, consistent with a role in germ plasm formation. Interestingly, overexpression of buc seems to generate ectopic germ cells in the zebrafish embryo. Because we discovered buc homologs in many vertebrate genomes, including mammals, these results identify buc as the first gene necessary and sufficient for germ plasm organization in vertebrates.

Circulating nucleic acids in plasma or serum.

BACKGROUND Nucleic acids can be found in small amounts in healthy and diseased human plasma/serum. Higher concentrations of DNA are present in the plasma of cancer patients sharing some characteristics with DNA of tumor cells. Together with decreased strand stability, the presence of specific oncogene or tumor-suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes may be detected. Moreover, tumor-related mRNA has been found circulating in the plasma/serum. CONCLUSIONS The results obtained in many different cancers have opened a new research area indicating that circulating nucleic acids might eventually be used for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer.

Alu Repeat Sequences Are Present in Increased Proportions Compared to a Unique Gene in Plasma/Serum DNA

Abstract: Small amounts of DNA circulate freely in plasma or serum, but the mechanism of release is not known. To determine if DNA is actively excreted from viable cells, we utilized real‐time PCR to measure the proportion of Alu repeat sequences compared to the β‐globin gene in serum and lymphocyte DNA in 27 cancer patients and 22 healthy controls. The proportion of Alu compared to β‐globin was significantly greater in serum DNA than in lymphocyte DNA both in control subjects (p= 0.003) and in cancer patients (p < 0.001). Overall, the proportion was similar in cancer and control patients (p= 0.79). Further experiments showed that the β‐globin gene was not more vulnerable to degradation by nuclease action than Alu sequences. Our results lead us to conclude that active DNA release is likely to play a significant role in the origin of circulating DNA.

Divergent effects of oxytocin treatment of obese diabetic mice on adiposity and diabetes

Oxytocin has been suggested as a novel therapeutic against obesity, because it induces weight loss and improves glucose tolerance in diet-induced obese rodents. A recent clinical pilot study confirmed the oxytocin-induced weight-reducing effect in obese nondiabetic subjects. Nevertheless, the mechanisms involved and the impact on the main comorbidity associated with obesity, type 2 diabetes, are unknown. Lean and ob/ob mice (model of obesity, hyperinsulinemia, and diabetes) were treated for 2 weeks with different doses of oxytocin, analogues with longer half-life (carbetocin) or higher oxytocin receptor specificity ([Thr4,Gly7]-oxytocin). Food and water intake, body weight, and glycemia were measured daily. Glucose, insulin, and pyruvate tolerance, body composition, several hormones, metabolites, gene expression, as well as enzyme activities were determined. Although no effect of oxytocin on the main parameters was observed in lean mice, the treatment dose-dependently reduced food intake and body weight gain in ob/ob animals. Carbetocin behaved similarly to oxytocin, whereas [Thr4,Gly7]-oxytocin (TGOT) and a low oxytocin dose decreased body weight gain without affecting food intake. The body weight gain-reducing effect was limited to the fat mass only, with decreased lipid uptake, lipogenesis, and inflammation, combined with increased futile cycling in abdominal adipose tissue. Surprisingly, oxytocin treatment of ob/ob mice was accompanied by a worsening of basal glycemia and glucose tolerance, likely due to increased corticosterone levels and stimulation of hepatic gluconeogenesis. These results impose careful selection of the conditions in which oxytocin treatment should be beneficial for obesity and its comorbidities, and their relevance for human pathology needs to be determined.

Leptin as a Potential Regulator of FGF21.

Background/Aims: Fibroblast growth factor 21 (FGF21), a potent metabolic regulator, has been shown to improve insulin sensitivity in animal models of insulin resistance. Several studies have focused on identifying mediators of FGF21 effects. However, the identification of factors involved in FGF21 regulation is far from complete. As leptin is a potent metabolic modulator as well, we aimed at characterizing whether leptin may regulate FGF21. Methods: We investigated a potential regulation of FGF21 by leptin in vivo in Wistar rats and in vitro using human derived hepatocarcinoma HepG2 cells. This model was chosen as the liver is considered the main FGF21 expression site. Results: We found that leptin injections increased plasma FGF21 levels in adult Wistar rats. This was confirmed in vitro, as leptin increased FGF21 expression in HepG2 cells. We also showed that the leptin effect on FGF21 expression was mediated by STAT3 activation in HepG2 cells. Conclusion: New findings regarding a leptin-STAT3-FGF21 axis were provided in this study, although investigating the exact mechanisms linking leptin and FGF21 are still needed. These results are of great interest in the context of identifying potential new clinical approaches to treat metabolic diseases associated with insulin resistance, such as obesity and type 2 diabetes.