Meeting EBTNA 9 ottobre 2015

 

 

Peter B. Gahan King’s College, London, UK .

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Components of the cytosolic and released virtosomes from stimulated and non-stimulated human human lymphocytes.

 

A number of early investigators demonstrated that both stimulated and non-stimulated lymphocytes released DNA (1-5). Subsequently, Stroun and Anker showed the released DNA to be newly synthesized with ³H-thymidine labeling studies (6-8). Furthermore, the DNA was associated with RNA (9). Since both nucleic acids were resistant to nuclease activity, it was considered that they were protected by lipoprotein. The presence of protein was identified when RNAse activity affected RNA only after a prior treatment with either pronase or proteinase k (9,10-13) while that of lipids was identified from the complex’s low density during upward sucrose density gradient centrifugation (10), freezing and thawing (10, 11) and the incorporation of radioactive phospholipid precursors (10,13,). Subsequent studies using radioactive precursors permitted the demonstration that the RNA, protein and associated phospholipids were (a) newly synthesized and (b) synthesized at about the same time (9-12). This DNA/RNA-lipoprotein complex, has an estimated size of ~5 x 10⁵ daltons (13) although the complex released from stimulated rat lymphocytes had a higher density than that released fom non-stimulated rat lymphocytes (10). The complex, termed a virtosome (14) is released in an apparently energy-dependent step (10), only from living cells (10,13,15-17) and in a controlled manner (18-21). Experiments employing radioactive precursors have shown that the DNA, RNA, phospholipid and proteins appear in the cytoplasm at about 3 h after commencing labeling and that the complex is released from cells 3 – 6 h later, depending on which cells were studied i.e. human, other mammalian, avian, amphibian and plant cells (1,6,8,10,15,16,20-22).

 

The complex does not appear to have a limiting membrane as shown by studies on the uptake and release of virtosomes between chick embryo fibroblasts (15) and on release from J774 cells and their uptake by non-stimulated lymphocytes (23).

 

Importantly, virtosomes released from one cell type can enter a different cell type resulting in a biological modification of the recipient cells e.g. transformation of NIH 3T3 cells on uptake of released mutant k-ras from SW480 cells (24), an allogenic T – B lymphocyte co-operation involving lymphocyte subsets from human donors with different allotypes (25,26) and DNA synthesis initiation in non-stimulated lymphocytes on uptake of virtosomes released by J774 and P497 tumour cells (23). Thus, the virtosome appears to be a novel cytoplasmic component that may act as an inter-cellular messenger.

 

However, the full structure of the complex has not been ascertained. In the present study, experiments were designed (a) to identify the lipids and proteins associated with both the cytosolic and released complexes, (b) the comparative amounts of proteins, lipids, DNA and RNA in cytosolic and released virtosomes and (c) the nature of the proteins present in the released virtosomes as opposed to those absent from the cytosolic virtosomes. However, as a first step to ensure that the virtosomes released from stimulated and non-stimulated lymphocytes were biologically active, the cytosolic and released virtosomes were isolated and tested for their biological activity, as previously described (23-26).

 

In addition to obtaining the overall content of DNA, RNA and phospholipids, the analysis of the individual phospholipids gave further confirmation for the absence of a classical membrane limiting the virtosome.