N-(4-hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid.
N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of particular clinical interest in cancer chemoprevention. We have examined the in vitro effects of HPR on lymphoid and myeloid malignant cell lines and found that at concentrations between 10(-5) and 3 x 10(-7) M it induces a dose-dependent growth inhibition (the peak plasma concentration in patients treated with HPR is 1 to 2 x 10(-6) M). The antiproliferative effect of HPR was, in all cell lines except K422, more potent than that induced by an equimolar dose of all-trans retinoic acid (RA). Also, this effect was irreversible on HL60 and DoHH2 cells that had been exposed to HPR (3 x 10(-6) M) for 24 h, but reversible on Raji and DHL4 exposed to the retinoid for 48 and 72 h, respectively. Time-course growth analysis showed that HPR at 3 x 10(-6) M or below induces a rapid fall of thymidine uptake and viability (> 90%), whereas between 10(-6) and 3 x 10(-7) M exhibits cytostatic effects. Interestingly, the RA-resistant HL-60R and NB306 cells, characterized by a point mutation in the retinoic acid receptor (RAR) and by the loss of the pml/RAR protein, respectively, were, like the parental RA-inducible HL-60 and NB4 cell lines, fully responsive to HPR, thereby suggesting that HPR and RA could act through different receptors or pathways. DNA flow-cytofluorimetric analysis revealed that HPR does not block cells in a specific phase of the cell cycle but triggers programmed cell death or apoptosis. This phenomenon was evidenced both by the visualization, on gel electrophoresis, of fragmented DNA, and by the "in cell" enzymatic labeling of DNA breaks with fluorescent dUTP. With the latter method, apoptotic cells become detectable by 6 h following exposure to 3 x 10(-6) M HPR. Ultrastructural examination of HPR-treated samples showed cells with chromatin compaction and cytoplasm condensation, characteristic of apoptotic cells. In conclusion, our study demonstrates that HPR suppresses malignant cell growth and induces apoptosis at pharmacologically relevant doses. The differential responsiveness by a number of cell lines, especially HL-60R and NB306, to HPR and RA indicates that these compounds may act through different receptors. The clinical use of HPR, particularly in retinoic acid-unresponsive acute promyelocytic leukemia patients, is suggested.
Delia
D
D
Division of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy.
Aiello
A
A
Lombardi
L
L
Pelicci
P G
PG
Grignani
F
F
Grignani
F
F
Formelli
F
F
Menard
S
S
Costa
A
A
Veronesi
U
U
D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000032:analysis / Q000378:metabolism; D004351:Drug Resistance; D017313:Fenretinide / Q000494:pharmacology*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000473:pathology; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D017313:Fenretinide; D014212:Tretinoin; D004247:DNA