Non-dividing Cell Virtosomes Affect In Vitro and In Vivo Tumour Cell Replication.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

The virtosome-a novel cytosolic informative entity and intercellular messenger.

Studies on a range of prokaryote and eukaryote cells and tissues have shown that a newly synthesized DNA/RNA-lipoprotein complex is released in a regulated manner. This complex, termed a virtosome, is a novel cytosolic component of eukaryote cells. The released virtosomes can readily enter other cells where they can modify the biology of the recipient cells. Such modifications include immunological changes and transformation from normal to cancer cells. The virtosomes form a normal component of the circulating nucleic acids in plasma and serum currently used for clinical diagnostic purposes. Given the transformative powers of virtosomes released from tumour cells, the presence of such a complex in human plasma could readily offer the basis of an alternative mechanism for the initiation of metastases.

Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia.

The analysis of total plasma DNA and the monitoring of leukemic clone-specific immunoglobulin and/or T-cell receptor gene rearrangements for the evaluation of minimal residual disease (MRD) in the plasma may be useful tools for prognostic purposes or for early detection of subclinical disease recurrence in children with acute lymphoblastic leukemia (ALL). The aim of this paper is to establish reference ranges for total plasma DNA concentrations and to test the feasibility of MRD measurements employing plasma DNA from children with ALL by using real-time quantitative (RQ)-PCR. Despite wide inter-individual variation, the median concentrations of total plasma DNA for 12 healthy donors (57 ng/ml), 21 children with ALL after day 4 of treatment initiation (62 ng/ml) and 13 children with other malignancies (76 ng/ml) were similar. However, ALL patients had significantly higher concentrations at diagnosis (277 ng/ml) and on treatment day 3 (248 ng/ml) before returning to normal afterwards. Early plasma DNA MRD kinetics could be established for 15 ALL patients and showed good concordance with bone marrow MRD. Plasma DNA was higher in children with ALL at diagnosis but returned to normal within the first four treatment days. Despite low concentrations of DNA, it is feasible to measure MRD kinetics in plasma DNA during ALL induction therapy by adapted real-time PCR methodologies.

A Preliminary Study of the Action of Virtosomes from Non-dividing Cells on Tumour Cell Replication in vitro and in vivo.

BACKGROUND: A DNA-RNA-lipoprotein complex, termed as virtosome, is released spontaneously from healthy human, other mammalian, avian, amphibian and plant cells in a regulated and energy-dependent manner. Studies on human and mouse lymphocytes, hepatocytes, NIH 3T3 cells and mouse tumour cell lines have shown that virtosomes may be acting as inter-cellular messengers. In particular, virtosomes from non-dividing cells blocked 3H-thymidine incorporation into DNA in tumour cell lines. OBJECTIVE: Study of the effect of virtosomes on tumors "in vivo" and "in vitro". METHODS AND RESULTS: The present study shows in vitro effects of virtosomes isolated from rat liver, essentially nondividing cell populations, on cultures of healthy smooth muscle cells (SMC), human umbilical vein endothelial cells (HUVEC), human fibroblasts (h-fibroblasts) and mouse embryonic fibroblasts (NIH-3T3) together with two tumour cell lines, human Duke's type B colorectal adenocarcinoma cells (SW480) and human connective tissue fibrosarcoma cells (HT1080). Multiplication of all cell lines was inhibited by the liver virtosomal preparation even with various dilutions of the extract (100 - 0.5%). In an in vivo study, tumours were initiated by subcutaneous injection of 1.0 x 106 DHD/K12-PROb cells in 6 weeks old male BIDX rats. Visible tumours (<1cm) appeared after 3 weeks and lung metastases after 8 weeks (80%). Virtosomes introduced via a tail vein on tumour initiation resulted in a reduction in tumour size and number. CONCLUSION: Virtosomal preparation from a non-dividing cell population inhibited cell division, reduced tumour size and partially blocked metastasis.

Prehistory of the notion of circulating nucleic acids in plasma/serum (CNAPS): birth of a hypothesis.

In the late 50s and early 60s of the last century, a theoretical fight was taking place between Western and Russian scientists about the theory explaining the mechanism of evolution. According to neo-Darwinism, evolution was the result of hazard and necessity, that is, mutations arriving by chance favoring the survival of the fittest. For the Russian geneticists, acquired characteristics were the basis of evolution, that is, the environment modified the characteristics of the gene. One of the main experiments on which the Russian geneticists based their theory was the transmission of hereditary characteristics by a special technique of grafting between two varieties of plants-a mentor plant and a pupil plant. The pupil variety being entirely dependent on the development of the mentor plant its hereditary characteristics were modified accordingly. In the Western world these experiments were regarded with doubt. We were among the few who tried to repeat this kind of experiment. After three generations of grafting between two varieties of eggplant, we succeeded in obtaining hereditary modifications of the pupil plants, which acquired some of the characteristics of the mentor variety. The linkage between some hereditary characteristics of the mentor plant were broken, the segregation of the offspring was abnormal, dominant characteristics appearing in the offspring of a recessive plant. Rather than adopting the views of the Russian scientists about acquired characteristics, we suggested that DNA was circulating between the mentor and pupil plants and assumed that some nucleic acid molecules bearing genetic information could enter the somatic and reproductive cells of the pupil plant at a propitious moment and remain active.

Immunological aspects of circulating DNA.

Nude mice were injected with DNA released by T lymphocytes previously exposed to inactivated herpes symplex type 1 or polio viruses. The serum of these mice was tested for its neutralizing activity. Injected nude mice synthesized antiherpetic or antipolio antibodies, depending on the antigen used to sensitize the T lymphocytes. Mice injected with DNA released by human T cells produced antibodies carrying human allotypes as they could be neutralized by antiallotype sera. However, mice that were injected with DNA released by antigen-stimulated murine T lymphocytes produced antiviral antibodies, which were not neutralized by anti-human allotype sera.

Transcession of DNA from bacteria to human cells in culture: a possible role in oncogenesis.

The human organism is continuously in close contact with microorganisms, especially bacteria. In the present work, by means of a real-time polymerase chain reaction (PCR) technique, we looked for the presence of a distinct bacterial gene in human cells. To this end, we cultured a human cell line, HL60, in a supernatant in which bacteria (Bacillus subtilis) had been grown. A transient transcession of bacterial DNA into the human cells was observed.

Circulating nucleic acids and evolution.

INTRODUCTION: J.B. Lamarck in 1809 was the first to present a theory of evolution. He proposed it was due to the adaptation of species to environmental changes, this adaptation being acquired by the offspring. In 1868, Darwin suggested that cells excrete gemmules, which circulate through the body and reach the gonads where they are transmitted to the next generation. His main argument came from graft hybrids. AREAS COVERED: In the fifties and sixties, Russian geneticists, rejecting neo-Darwinism, said that acquired characteristics were the basis of evolution. The main experiments on which they based their theory were the transmission of hereditary characteristics by a special technique of grafting between two varieties of plants. We repeated this kind of experiment and also succeeded in obtaining hereditary modifications of the pupil plants that acquired some characteristics of the mentor variety. Rather than adopting the views of the Russian scientists, we suggested that DNA was circulating between the mentor and pupil plants. Hirata's group have shown recently, by using molecular techniques such as cloning, RFLP PCR and sequencing some genes of their graft hybrids of pepper plants, that transfer of informative molecules from the mentor to the pupil plant does exist. Nucleic acids are actively released by cells; they circulate in the body. They can transform oncogenically or trigger antibody response but the only genetic transformation showing that DNA can go from the soma to the germen comes from graft hybrids. EXPERT OPINION: This suggests that circulating nucleic acids, in this case DNA, like Darwin's gemmules, play a role in the mechanism of evolution.

Cell-free nucleic acids circulating in the plasma of colorectal cancer patients induce the oncogenic transformation of susceptible cultured cells.

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the development of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative phenomenon is tentatively referred to as "genometastasis." In the present study, we examined the effects on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from patients with K-ras-mutated colorectal tumors or from healthy subjects using two different protocols: direct addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and cells, which were separated by a membrane with 0.4-mum pores. In plasma-treated hASCs, no K-ras-mutated sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells (murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences, p53 sequences, and beta-globin-encoding sequences. Moreover, NIH-3T3 cells that had been cultured with plasma from patients with colon cancer were oncogenically transformed, as shown by the development of carcinomas in nonobese diabetic-severe combined immunodeficient mice after the injection of such cells. Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete absence of cells in the plasma. We only observed noncell particles, having diameters of <0.4 mum. Our results indicate that plasma from cancer patients is able to transform cultured cells oncogenically, supporting the previously proposed hypothesis of genometastasis.

Metabolic DNA as the origin of spontaneously released DNA?

A DNA fraction is spontaneously released from living, but not dead or dying, human, other mammalian, avian, amphibian, plant, and prokaryote cells. The spontaneously released DNA fraction has been shown to be (a) present in both actively dividing and nondividing, differentiated cell populations; (b) labile; (c) associated with DNA-dependent RNA or DNA polymerase; (d) associated with an RNA fraction; and to have (e) a lower molecular weight than the typical genetic DNA fraction; and (f) Alu repeat sequences in increased proportions compared to a unique gene in plasma/serum. On the other hand, early autoradiographic and biochemical and quantitative cytochemical and cytophysical studies on DNA permitted the identification of a DNA fraction which was (1) present in both actively dividing and nondividing, differentiated cell populations; (2) labile; and (3) had a lower molecular weight than the typical genetic DNA fraction. This DNA fraction was termed metabolic DNA (m-DNA) and was proposed as possibly forming extra gene copies for the rapid production of m-RNA, to be destroyed subsequently. Therefore, we suggest that the metabolic DNA fraction might represent the precursor to the formation of the spontaneously released DNA fraction.

Plasma DNA microsatellite panel as sensitive and tumor-specific marker in lung cancer patients.

The majority of lung cancer patients have tumor-derived genetic alterations in circulating plasma DNA that could be exploited as a diagnostic tool. We used fluorescent microsatellite analysis to detect alterations in plasma and tumor DNA in 34 patients who underwent bronchoscopy for lung cancer, including 11 small cell lung cancer (SCLC) and 23 nonsmall cell lung cancer (NSCLC) (12 adenocarcinomas, 11 squamous cell carcinomas) and 20 controls. Allelotyping was performed with a selected panel of 12 microsatellites from 9 chromosomal regions 3p21, 3p24, 5q, 9p, 9q, 13q, 17p, 17q and 20q. Plasma DNA allelic imbalance (AI) was found in 88% (30 of 34 patients), with a similar sensitivity in SCLC and NSCLC. In the 24 paired available tumor tissues, 83% (20 of 24) presented at least 1 AI. Among these patients, 85% (17 of 20) presented also at least 1 AI in paired plasma DNA, but the location of the allelic alterations in paired plasma and tumor DNA could differ, suggesting the presence of heterogeneous tumor clones. None of the 20 controls displayed plasma or bronchial DNA alteration. A reduced panel of six markers (at 3p, 5q, 9p, 9q) showed a sensitivity of 85%. Moreover, a different panel of microsatellites at 3p and 17p13 in SCLC and at 5q, 9p, 9q and 20q in NSCLC patients could be specifically used. Analysis of plasma DNA using this targeted panel could be a valuable noninvasive test and a useful tool to monitor disease progression without assessing the tumor.