Journal Article

Apoptosis of squamous cells at different stages of carcinogenesis following 4-HPR treatment

Silvia Bruno, Claudya Tenca, Daniele Saverino, Ermanno Ciccone and Carlo E. Grossi

in Carcinogenesis

Volume 23, issue 3, pages 447-456
Published in print March 2002 | ISSN: 0143-3334
Published online March 2002 | e-ISSN: 1460-2180 | DOI:
Apoptosis of squamous cells at different stages of carcinogenesis following 4-HPR treatment

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Squamous cell carcinoma (SCC) is the end product of a multistep process characterized by a progression from normal epithelial cells through metaplastic or dysplastic intraepithelial changes that evolve into invasive cancer. Since retinamides have shown promising in vivo anti-tumoral activity, we studied effects and effector mechanisms of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) on squamous cells at progressing stages of tumorigenesis. To this end, an in vitro model of squamous carcinogenesis consisting of normal human keratinocytes, human papilloma virus (HPV)-immortalized keratinocytes (UP) and tumorigenic HPV-immortalized/v-Ha-ras transfected keratinocytes (UPR) was used. 4-HPR treatment affected cell growth at doses higher than 1.5 μM. Flow cytometric measurements of DNA content and annexin V revealed that cell growth decrease was mainly due to apoptosis at 4-HPR concentrations of or below 15 μM, and necrosis at higher concentrations. The effects were similar in the three cell types of the in vitro model, as well as in three SCC cell lines, suggesting that sensitivity to 4-HPR is independent of the degree of squamous cell tumorigenesis in the in vitro model. We further investigated whether mitochondrial damage was involved in the course of 4-HPR-induced apoptosis. Treatment of squamous cells with the antioxidant L-ascorbic acid inhibited apoptosis, indicating that 4-HPR increases production of free radicals. Measures of mitochondrial membrane potentials showed that 4-HPR induced membrane permeability transition (MPT), and that MPT-inhibitors were able to reduce apoptosis. This indicates that MPT is involved in apoptosis signalling by 4-HPR. Finally, we studied the role of caspases. We found that caspases 8, 9 and 3 participate in 4-HPR-mediated apoptosis of squamous cells, and that MPT is an upstream event that regulates caspase activity. Caspase 8 was activated independently of the Fas–Fas ligand pathway.

Keywords: 4-HPR, N-(4-hydroxyphenyl)retinamide;; DiOC6, 3,3′-dihexyloxacarbocyanine iodide;; HPV, human papilloma virus;; mAb, monoclonal antibody;; MPT, mitochondrial permeability transition;; PI, propidium iodide;; ROS, reactive oxygen species;; SCC, squamous cell carcinoma;; TPCK, N-tosyl-l-phenylalanyl chloromethylketone.

Journal Article.  6950 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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