The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) can inhibit the growth and induce apoptosis of tumor cells. In this study we analysed the growth suppressive e ect of HPR on human breast cancer cell lines in vitro and the role of the retinoblastoma protein (pRb) in this response. Treatment of MCF7, T47D and SKBR3 for 24 ± 48 h with 3 mM HPR, a concentration attainable in vivo, resulted in growth inhibition and marked dephosphorylation of pRb involving Ser 612 , Thr 821 , Ser 795 and Ser 780 , target residues for cyclin-dependent kinase 2 (Cdk2) the former two, and Cdk4 the latter two. Interestingly, this dephosphorylation of pRb occurred in S-G2-M phase cells, as revealed by experiments on cells fractionated by FACS according to the cell cycle phase, hence suggesting that the retinoid interferes with the regulation of pRb phosphorylation. The in vitro phosphorylation of a GST-pRb recombinant substrate by Cdk2 immunocomplexes from MCF7, T47D and SKBR3 was markedly suppressed after HPR treatment, whereas that by Cdk4 complexes was suppressed in T47D and SKBR3 but not in MCF7. The steady-state levels of Cdk2, Cdk4 and Cyclin A proteins were una ected by HPR, while those of Cyclin D1 were signi®cantly reduced in all three cell lines. Interestingly, Cyclin D1 downregulation by HPR correlated with transcriptional repression, but not with enhanced proteolysis of Cyclin D1 typically elicited by other retinoids. Collectively, our data suggest that the antiproliferative activity of HPR arises from its capacity to maintain pRb in a de-phosphorylated growthsuppressive status in S-G2/M, possibly through Cyclin D1 downregulation and inhibition of pRb-targeting Cdks.

N‐(4‐hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of clinical use in breast cancer chemoprevention. Its in vitro effects on tumor cell lines include growth inhibition and apoptosis and are, to a large extent, independent of the activation of retinoic acid receptors. We have performed a cDNA differential display analysis to identify genes potentially relevant for understanding the mechanism of action of HPR. We report that prosaposin (PSAP), a gene encoding for the common precursor protein of saposins (cofactors involved in the metabolism of various sphingolipids), was significantly up‐regulated, both at transcriptional and protein level, in MCF7 and T47D breast cancer cell lines after treatment with HPR, but not with all trans retinoic acid (ATRA). Remarkably, this response was also induced by oxidants, whereas it was abrogated by antioxidants in cells treated with HPR, which implies a direct role of free radicals generated by HPR in prosaposin up‐regulation. Stable transfectants of T47D cells overexpressing prosaposin (T47D‐PSAP) exhibited morphological changes, reduced proliferation, and impaired attachment to culture plates, which correlated with down‐regulation of α6, β1, and β4 subunits of the integrin family of adhesion receptors. Concordant with a role of prosaposin in ceramide generation, increased amounts of this lipid were found in T47D‐PSAP and in HPR‐treated T47D cells. Moreover, treatment of T47D with C2‐ceramide resulted in the up‐regulation of prosaposin and down‐regulation of integrin receptors. Altogether, these findings establish a link between prosaposin, ceramide, and integrin receptor regulation. In view of the association between integrin down‐regulation and suppression of metastatic capacity, our data raise the possibility that the cancer chemopreventive activity of HPR may rely, at least in part, on its capacity to regulate integrin receptors through prosaposin.

Summary ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1-32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other ~70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40-50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway.

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We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electrophoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large-scale population studies because of the automation and speed of capillary electrophoresis.