Specific lipoplex-mediated antisense against Bcl-2 in breast cancer cells: a comparison between different formulations.

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3ss[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms--either large unilamellar vesicles (approximately 100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.

P53 in cytoplasm may enhance the accuracy of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The tumor suppressor protein p53 displays 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 is responsible for the conversion of the viral genomic ssRNA into the proviral DNA in the cytoplasm. The relatively low fidelity of HIV-1 RT was implicated as a dominant factor contributing to the genetic variability of the virus. The lack of intrinsic 3' --> 5' exonuclease activity, the formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be determinants for the low fidelity of HIV-1 RT. It was of interest to analyse whether the cytoplasmic proteins may affect the accuracy of DNA synthesis by RT. We investigated the fidelity of DNA synthesis by HIV-1 RT with and without exonucleolytic proofreading provided by cytoplasmic fraction of LCC2 cells expressing high level of wild-type functional p53. Two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of noncomplementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of cytoplasmic fraction of LCC2 cells with both RNA/DNA and DNA/DNA template-primers with the same target sequence. The mispair extension frequencies obtained with the HIV-1 RT in the presence of cytoplasmic fraction of LCC2 cells were significantly lower (about 2.8-15-fold) than those detected with the purified enzyme. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53 in cytoplasm) activities was observed; p53 preferentially hydrolyses mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. The data suggest that p53 in cytoplasm may affect the accuracy of DNA replication and the mutation spectra of HIV-1 RT by acting as an external proofreader. Furthermore, the decrease in error-prone DNA synthesis with RT in the presence of external exonuclease, provided by cytoplasmic p53, may partially account for lower mutation rate of HIV-1 observed in vivo.

p53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells.

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.