In BALB/c-3T3 cells, expression of the c-myc gene is stimulated by platelet-derived growth factor (PDGF). Using mouse mammary tumour virus promoter: c-myc recombinant plasmids, 3T3 sublines were constructed in which hydrocortisone was the primary determinant of myc mRNA content. The c-myc gene product is an intracellular mediator of the growth response to PDGF though probably not the only one. Both the human and the mouse c-myc genes stimulate clonal growth of 3T3 cells in PDGF-free medium suggesting new strategies for analysis of oncogenes which do not function in focus formation assays.

The interaction of small cationic vesicles composed of dioctadecyldimethylammonium bromide (DODAB) with normal versus transformed mouse fibroblasts is described using cell microelectrophoresis, turbidimetry, and cell viability assays over a wide range of DODAB concentrations (10-7−10-3 M). Normal and transformed cells (104 cells/mL) attain a point of zero charge at, respectively, 18.0 and 1.6 μM DODAB. Further increasing DODAB concentration (C) generates positively charged cells. At 105 cells/mL and C ≥ 50 μM, DODAB induces cell−cell adhesion. For transformed and normal cells, peak adhesion occurs at 100 and 1000 μM DODAB, respectively. Upon 0.5 h interaction time with 100 μM DODAB, at 104 cells/mL, 20% of cell death is obtained for normal cells whereas transformed cells remain unaffected. Transformed cells have a higher affinity for DODAB vesicles than their normal counterparts but are more resistant to DODAB-induced cell death. The results indicate that DODAB vesicles interact with cells with very high affinity at low ionic strength and are not toxic below 1 mM, suggesting that they might successfully deliver oppositely charged proteins or DNA strands to cells. These results may be of importance for liposome-mediated processes currently being used for drug or gene delivery to cells.

No abstract

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factorsinduced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a speci®c P13K inhibitor) leads to decreased AP-1 activity. Supershift and Western blot analysis with speci®c antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results con®rm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be su cient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD-induced cytotoxicity and cell transformation.