No abstract

, a cyclin-dependent kinase inhibitor, may act as an antioncogene, but may also behave as a tumor promoting factor by inhibiting apoptosis. p21 is also a transcriptional regulator, exerting this activity independently of cyclin-dependent kinases. Increased p21 protein levels were found in a subset of melanomas. However, the mechanism(s) contributing to the tolerance of high p21 levels in melanoma help explain the tolerance of increased p21 levels found in some melanomas.

Microphthalmia-associated transcription factor (MITF) activates the expression of melanocyte-specific markers and promotes the survival of embryonic, adult and malignant melanocytes. Although numerous MITF-dependent downstream genes have been identified, the mechanisms by which the MITF activity is coregulated remain elusive. Here we used a non-melanocytic cell line U2-OS as a model in which MITF evokes transcription of a paradigmatic MITF target tyrosinase and show that the adenoviral E1A protein represses the MITF-driven transcription in these cells. The E1A CR1 domain (which alone is insufficient to bind p300) was sufficient for repression, while the N-terminus, through which E1A binds the p300/CBP proteins and other coactivators, was unable to repress. Correspondingly, CR1 inhibited colony formation of MITF-positive, but not MITF-negative, melanoma cells. The repression by CR1 was largely independent of the PCAF-binding motif, previously recognized to be necessary for suppression of muscle-specific enhancer. Interestingly, CR1 conferred transcriptional competence to the MITF-CR1 chimera in which the MITF portion was rendered transcription-deficient. Moreover, MITF mutants defective in binding to p300/CBP in vivo still activated transcription, further supporting a p300/CBP-independent coactivation of MITF targets. MITF is amplified in a subset of melanomas and is thought to be required for sustained proliferation of malignant melanocytes. Our results suggest that understanding how CR1 represses Mitf activity may reveal a route to melanoma therapy.

Purpose Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO 2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO 2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO 2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO 2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO 2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods We determined the size and crystallinity of TiO 2 P25. We used four techniques for TiO 2 P25 dispersion. We estimated the colloid stability of TiO 2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO 2 P25 (0–100 µg.mL −1 ) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO 2 P25. Under these conditions, TiO 2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO 2 P25 in A549 cells were met, similar results on the biological effects of TiO 2 P25 were obtained in two independent cell laboratories. Conclusion We optimized the experimental conditions of TiO 2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO 2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO 2 P25 as a reference material.