In the ovarian adenocarcinoma subline N.I, all-trans retinoic acid (ATRA) induced substantial cell death. This response was elicited only at decreased serum levels. Exposure of N.I cells to increasing concentrations of ATRA was accompanied by a considerable up-regulation of c-myc transcript levels that correlated with the rate of cell killing, which itself was an active process as judged by sustained transcriptional expression. ATRA-triggered rounding and detaching of single cells from the substratum was accompanied by degradation of genomic DNA. We show that the N.I model cell line, which is otherwise highly ATRA-resistant, can undergo an ATRA-triggered suicide program when serum is limited. The accompanying c-myc up-regulation seems to be mediated by retinoic-acid-receptor-independent pathways involving membrane-associated phospholipases instead, because manoalide partly suppressed c-myc induction by ATRA but left constitutive c-myc expression unaffected.

The responsiveness of estrogen receptor (ER)-positive breast cancer to endocrine therapy is frequently reduced in cells over-expressing c-erbB-2. Stimulation of ER suppresses c-erbB-2, indicating that estrogen controls the activity of c-erbB-2. Heregulin (HRG) has been described to bind to c-erbB-3/c-erbB-4 and to stimulate c-erbB-2. Here we describe the effects of HRG on cell growth and on ER and c-erbB-2 expression in breast cancer cell lines containing distinct levels of c-erbB-2 and ER (BT-474: c-erbB-2 , ER+; MDA-MB-361: c-erbB-2++, ER++; MCF-7: c-erbB-2+, ER ). Proliferation of estrogen-stimulated, c-erbB-2 and ER-positive cells is inhibited by HRG in a dose-dependent manner. In addition, HRG dose-dependently inhibits ER expression. Estrogen, however, inhibits c-erbB-2. Estrogen-mediated down-regulation of c-erbB-2 is most pronounced in MCF-7 but weaker in BT-474. In the latter cells HRG efficiently blocks the estrogenic effect on c-erbB-2. In MCF-7 cells, however, the inhibition of c-erbB-2 cannot be completely reverted by HRG. This modulation occurs in all 3 cell lines at protein, RNA and transcriptional levels, suggesting that the activity of the c-erbB-2 promoter, which contains an estrogen-responsive region, is affected by HRG. The intensity of the mutual inhibition between the HRG/c-erbB-2 and the estrogen/ER system depends on the relative levels of ER and c-erbB-2 expression in the respective cell lines.

Summary We investigated the effects of all-trans retinoic acid (ATRA) and fenretinide (4-HPR) on c-erbB-2 expression in SK-BR-3, and MCF-7 breast cancer cells and on the growth, differentiation, apoptosis and cisplatin (CDDP) sensitivity of SK-BR-3 cells. It has been reported that oestrogen inhibits c-erbB-2 in oestrogen receptor-positive breast cancer cells. Using ELISA, Westem and Northem analysis we have demonstrated that ATRA and 4-HPR exert similar effects down-regulating c-erbB-2 protein and mRNA in c-erbB-2-overexpressing SK-BR-3 and BT-474 and in normally expressing MCF-7 cells. Both retinoids inhibit SK-BR-3 cell growth. ATRA induces cellular enlargement and flattening, suggesting epithelial differentiation. 4-HPR causes nuclear and cytoplasmic condensation, DNA fragmentation and externalization of phosphabdylserine, indicating apoptosis. c-erbB-2 expression/activity has been linked to sensitivity against CDDP. Therefore, combinations of ATRA or 4-HPR with CDDP were tested for their anti-proliferative activity. Retinoid-conditioned cells were either exposed to retinoid and CDDP (schedule 1, 'continuous retinoid treatment') or to CDDP alone (schedule II, 'retinoid pretreatment'). This retinoid-conditioning followed by CDDP ± retinoid yields stronger growth inhibition compared with unconditioned cells, which were exposed to CDDP ± retinoid (schedule 111, 'no retnoid pretreatment'). The inefficacy of schedule Ill indicates that retioidconditioning is essential for the improvement of the antiproliferative effect. The interactions in schedules I and II are synergistic for ATRA and CDDP, but slightly antagonistic for 4-HPR and CDDP. However, 4-HPR + CDDP is more effective in growth inhibition than each drug alone.

Sodium butyrate (NaB), a physiologically produced short chain fatty acid, dramatically changes the growth rate and also the morphology of a fast growing subclone (N.1) derived from the heterogenous human ovarian carcinoma HOC-7. The mRNA of the growth related proto-oncogene c-myc, constitutively expressed in N.1 cells decreased significantly within 24 h of NaB treatment and remained suppressed until the NaB block was released. Down-regulation was accomplished partially by accelerating degradation of c-myc mRNA and by inhibiting splicing of c-myc transcripts. We demonstrated that NaB blocked general mechanisms in signal transduction, such as the release of Ca2+ from intracellular stores, and modulated the activity of serine/threonine kinases. The multiple effects of sodium butyrate on HOC-7 derivatives, as well as on a variety of other cell types investigated by others, may be due to interference with general mechanisms of signal transduction.

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