PPARalpha and PPARgamma are co-expressed, functional and show positive interactions in the rat urinary bladder urothelium.

Some dual-acting PPARalpha + gamma agonists cause cancer in the rat urinary bladder, in some cases overrepresented in males, by a mechanism suggested to involve chronic stimulation of PPARalpha and PPARgamma, i.e. exaggerated pharmacology. By western blotting, we found that the rat urinary bladder urothelium expressed PPARalpha at higher levels than the liver and heart, and comparable to kidney. Urothelial expression of PPARgamma was above that of fat, heart, skeletal muscle and kidney. Male rats exhibited a higher PPARalpha/PPARgamma expression balance in the bladder urothelium than did female rats. Rats were treated by gastric gavage with rosiglitazone (PPARgamma agonist), fenofibrate (PPARalpha agonist) or a combination of rosiglitazone and fenofibrate for 7 days. In the urothelium, the transcription factor Egr-1 was induced to significantly higher levels in rats co-administered rosiglitazone and fenofibrate than in rats administered either rosiglitazone or fenofibrate alone. Egr-1 was also induced in the heart and liver of rats treated with fenofibrate, but a positive interaction between rosiglitazone and fenofibrate with regards to Egr-1 induction was only seen in the urothelium. Thus, in the rat urinary bladder urothelium, PPARalpha and PPARgamma were expressed at high levels, were functional and exhibited positive interactions. Interestingly, fenofibrate induced the peroxisome membrane protein PMP70 not only in liver, but also in the bladder urothelium, opening the possibility that oxidative stress may contribute to rat urothelial carcinogenesis by dual-acting PPARalpha + gamma agonists.

Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression.

Insulin-exposed rat mammary cancer cells were flow sorted based on a c-myc reporter plasmid encoding a destabilized green fluorescent protein. Sorted cells exhibited gradual increases in c-myc levels. Cells overexpressing c-myc by only 10% exhibited phenotypic changes attributable to c-myc overexpression, such as cell cycle disturbances, increased cell size, and overexpression of the S6 ribosomal protein. Cells overexpressing c-myc by 70% exhibited additional phenotypic changes typical of c-myc overexpression, such as increased histone H3 phosphorylation, and reduced adherence. Sorted cells also exhibited overexpression of the IGF-1R, and slightly elevated expression of the IR. Increased susceptibility to the mitogenic effect of insulin was seen in a small proportion of the sorted cells, and insulin was more effective in activating the p44/42 MAPK pathway, but not the PI3K pathway, in the sorted cells than in the nonsorted cell population. To our knowledge, this is the first in vitro system allowing functional coupling between mitogenic signaling by a well-defined growth factor and gradual overexpression of the normal, endogenous c-myc gene. Thus, our flow-sorting approach provides an alternative modeling of the receptor-mediated carcinogenic process, compared to the currently used approaches of recombinant constitutive or conditional overexpression of oncogenic transmembrane receptor tyrosine kinases or oncogenic transcription factors.

MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface.

Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF-7 assay. The optimized MCF-7 assay described here is of relevance for in vitro toxicological testing and carcinogenicity safety assessment of new insulin compounds.