Latest Exposé on the Fluid Genome
Russian scientists suggest that circulating DNA from dead cells are taken in by living cells in order to replace mutated genes with good copies. What are the implications for the safety of transgenic DNA? Dr. Mae-Wan Ho reports.
Geneticists have recently made a surprising discovery. Substantial amounts of degraded genomic DNA are present in the in blood plasma and fluids surrounding cells, coming from cells in the body that have died. This circulating DNA binds to receptors on the surface of living cells and is taken up and transported to the cell nucleus.
Now, a group of scientists in Russia from the Novosibirsk Institute of Bioorganic Chemistry and the Institute of Cytology and Genetics, Russian Academy of Sciences, Siberia, have taken this finding further.
They suggest that, within the nucleus of the cell, homologous recombination of genomic DNA with these fragments takes place, and is initiated by the fragments. Homologous recombination is the exchange of parts between DNA sequences that are very similar or nearly identical. They suggest that this process can correct mutations as well as induce genetic changes, with the external DNA fragments serving as reference molecules [1]. This would ensure that all the cells in the body have the same genome.
So, provided the reference DNA itself does not contain harmful mutations, it would be useful for eliminating mutations from the cells and keeping the body healthy.
Cancer is a multifactorial genetic disease and numerous genetic changes, both mutational and inherited, are thought to be responsible. If malignant cells are offered genomic DNA from a healthy subject as external reference, couldn’t the reverse transformation of malignant to normal cells take place?
They tested the idea in three cultured human cell lines, derived from breast cancer cells (BT474), ovarian carcinoma cells (OVCAR5), and leukemic cells (HL60). These were treated either with chromatin (complex of DNA and protein) from human sperm of healthy subjects, digested with enzymes to obtain fragments 200 to 3000 bp; or else with genomic DNA isolated from sperm cells with a commercial reagent kit.
They did find changes in expression of some proteins associated with cancer, scrambling of the cell genome due to the DNA introduced (see box), as well as changes in the behaviour of the cells.
In each experiment, 2ug DNA was added to each well of 100 000 cells, and refreshed every three days for two weeks. Controls got no DNA.
After the experiment, the cells were analysed for ‘cancer-specific antigens’ (taken to be diagnostic of cancer). The results showed a significant decrease in the expression of erb-B2 (a protein of the epidermal growth factor receptor) in the OVCAR5 and BT474 cells. In OVCAR5 and HL60, the same treatments led to significant decrease in the content of cyclin D1 protein, whose expression is often correlated with rapid cell division.
Chromatin gave more decrease in cancer-specific antigens than DNA. Genomic fingerprinting with random amplification of polymorphic DNA demonstrated differences between treated and control samples, indicating that the effect of the DNA preparation was due to multiple genomic rearrangements.
The DNA therapy caused no toxic effects or changes in cell protein expression for the first week. After two weeks, however, the cells noticeably changed. On the 15th to 16 day, cell numbers decreased. In the case of the Bt474 cells, the number of cells was only half that of the control without DNA, and a proportion of the cells were dead. These changes were irreversible even if no further DNA preparations were added. Cell growth was further inhibited and stopped even upon treatment with purified DNA instead of chromatin. After transferring the cells to a fresh plate, the cells attached to plastic and remained alive for a month, though they did not form a complete monolayer and their number decreased constantly. So, both malignant cell death and arrest of cell division were observed.
There is no doubt that the DNA had affected the cells, but it is not clear whether the effects can be regarded as a ‘cure’ of the cancer, or whether they had anything to do with the DNA being taken up by the cells.
In the early 1950s, British biomedical researcher Peter Medawar and his team reported that mice tolerant to a foreign graft could be obtained by prenatal injection of donor blood lymphocytes into recipient mice before giving them the graft. The question as to why antigenic challenge can be either tolerated or immunogenic (provoking an immune reaction) is still up in the air.
Another phenomenon that remains unexplained is the inheritance of somatically acquired tolerance, as reported by immunologists Gorczinsky and Steele in the 1980s. This meant that the state of immunological tolerance, once acquired, could be passed on to the offspring, not just by a female, but by a male as well. That is because, as Gorczinsky and Steele claimed, some change in the DNA is involved. This aroused so much controversy that Nature’s then Editor, John Maddox declared Steele’s book [2] should be ‘burnt’ for proposing the Lamarckian heresy. And Medawar and others published articles in the journal refuting their work.
Lamarck, and not Darwin, was the first to propose the theory of evolution in the early 1800s. Unfortunately, Lamarck was ridiculed by Darwin’s followers - neo-Darwinians - for believing in the inheritance of acquired characters, which, to this day, is attributed to Lamarck. In reality, Darwin himself was also a ‘Lamarckian’. Furthermore, all the findings in genetics since the mid-1970s has vindicated Lamarck’s major thesis that there was much more feedback between the environment and the genome than hitherto supposed [3].
So, could it be that immunological tolerance was linked to the genomic DNA injected with the lymphocytes that converted genes in the graft-recipient’s cells?
The Russian researchers carried out another experiment. DNA from mice prone to develop tumours, were injected into another strain that was resistant to tumour cells from the first strain. After the injection, 30 to 50% of the resistant mice developed tumours at the site of injection, whereas none of the control, non-injected, mice developed any tumours. In other words, the resistant mice had become tolerant to the tumour cells.
No DNA data were provided, which would have made the results more convincing. If they are right, it would suggest that horizontal transfer of genetic material is a normal physiological process between cell communities, which include the organism’s own DNA as well as introduced, foreign DNA. This has significant implications for the safety of transgenic DNA in food or taken in as vaccines, as we have warned [4].
Three years ago, another research group had reported that DNA from dead cells is scavenged by living cells [5], and that integrated viral sequences may be preferentially iincorporated into genomes, with potentially harmful effects including cancer. In our report, we have warned against the use of viral sequences in transgenic constructs, such as the cauliflower mosaic viral promoter now found to have widely contaminated landraces of maize in Mexico [6].
The genetic material of dead cells is scavenged by other cells. It is taken up by phagocytosis – a kind of eating response in which the cell envelops the material – and is then either metabolised to generate energy and raw materials for building the cell, or it may be incorporated into the genome of the cell. Integrated viral sequences may be preferentially incorporated.
Researchers used DNA from killed human lymphoid cell lines with integrated Epstein-Barr virus (EBV) as marker to follow the fate and expression pattern of the DNA taken up by various other cell lines. The lymphoid cells were killed by an irradiation procedure or a drug that fragmented DNA, and then added to cultures of human fibroblasts, macrophages, or bovine aortic endothelial cells. They found that all the living cells took up the DNA from the killed lymphoid cells, but only DNA from lymphoid cell lines with integrated EBV resulted in expression of viral genes and incorporation of DNA containing viral sequences into the cells’ genome. DNA from killed lymphoid cell-lines with non-integrated EBV (existing as episomes) did not result in expression of viral genes, suggesting that viral sequences were not incorporated into the living cells’ genome.
The researchers also found that the frequency of horizontal transfer of human DNA to the genome of bovine cells was greatly increased in DNA from lymphoid lines with integrated EBV compared with the same lymphoid lines without any integrated EBV; furthermore, almost all of the transferred DNA was associated with the integrated EBV. It suggests that the integrated EBV may be preferentially transferred and incorporated. The authors conclude, "we speculate that similar mechanisms of horizontal DNA transfer may be of importance in conditions characterized by high levels of apoptosis [ie, cell death], eg, in tumours treated with irradiation or chemotherapy".
Ref. Holmgren, L., Szeles, A., Rajnavolgyi, E., Foldman, J., Klein, G., Ernberg, I. and Falk, K.I. (1999). Horizontal transfer of DNA by the uptake of apoptotic bodies. Blood 93, 3956-63.
Our comment: The results suggest that integrated viral sequences may be more invasive than other parts of the genetic material. This is in line with our suggestion that transgenic DNA may be more prone to transfer horizontally (see Viral Gene Switch – A Recipe for Disaster? This issue). Also, the authors’ speculation that similar mechanisms of horizontal DNA transfer may occur in tumours treated with irradiation or chemotherapy raises questions on the possibility that such treatments may spread cancer to other cells, assuming that the EBV is an important causal agent of tumour-formation. (M.W.H.)
Article first published 02/05/02
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