The study of the mechanism of neoplastic transformation induced by dnase i encapsulated in liposomes
Baltimore, Md., Johns Hopkins Univ., Diss., 1982
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| Sprache: | eng |
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Tag hinzufügen | Zusammenfassung: | Baltimore, Md., Johns Hopkins Univ., Diss., 1982 To study somatic mutation and neoplastic transformation (NT) in diploid Syrian hamster embryo (SHE) cells, liposomes were used to deliver active DNase I inside the cells, thus providing a direct and specific perturbation to the genetic apparatus. This study has shown (1) the entrance of DNase I inside the cell by dose dependent cytotoxicity (2) the entrance of DNase I into nucleus by the induction of mutation at the HGPRT locus and chromosomal aberrations. By the use of trace amounts of ('14)C Carbonic anhydrase, it was shown that 2-2.6% of enzyme was entrapped in liposomes, of which (TURN)3% could enter SHE cells, introducing 10('7) molecules per cell. For NT studies SHE cells were treated with various doses of DNase I-in-liposomes (DL) and appropriate controls. Within 2-3 weeks cells entered a 'crisis' state. 32 out of 34 control cultures senesced. All eight independently DL treated cultures established immortal lines. The DL treated cultures showed growth characteristics distinct from the two control cultures. The DL cultures showed: (1) colony formation at low cell density, (2) increased saturation density, (3) growth in 1% serum medium, (4) growth in 0.3% agar and (5) tumorgenicity in 100% of injected animals. DL treated cultures started to grow in 1% serum after 41-161 population doublings (PD) and in 0.3% agar after 39-57 PD. The PD required for the exponential growth in agar was proportional to the initial concentration of DNase I. The comparison of the temporal acquisition of both phenotypes indicates that they were acquired independently and that 1% serum growth is not a prerequisite for anchorage independent phenotype. This novel approach indicates that damage to DNA alone can initiate NT. The detection of mutation at the HGPRT but not ouabain locus, and induction of chromosomal aberrations suggest that the initial DNase I insult resulting in NT may be due to chromosomal rather than point mutation. The specific damage to DNA, however, is not sufficient for NT since a long progression process is required after the initial insult. To determine if epigenetic factors play a role in NT, this new technique could be used for delivery of RNases or proteases inside cells. This finding also implicates the possibility of initiation of NT due to insults causing the release of internal nucleases. |
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| Beschreibung: | 260 S. |