Most breast carcinomas that are estrogen receptor (ER) and progesterone receptor (PR) positive respond initially to an endocrine therapy, but over time, they develop resistance (acquired hormone resistance). Others, however, fail to respond from the beginning (constitutive resistance). Overcoming hormone resistance is one of the major desirable aims in breast cancer treatment. Using the medroxyprogesterone acetate (MPA)-induced breast cancer mouse model, we have previously demonstrated that antiprogestin-responsive tumors show a higher expression level of PR isoform A (PRA) than PR isoform B (PRB), while tumors with constitutive or acquired resistance show a higher expression level of PRB. The aim of this study was to investigate whether PRA silencing in resistant tumors was due to PRA methylation. The CpG islands located in the PRA promoter and the first exon were studied by methylation-specific PCR (MSP) in six different tumors: two antiprogestin-responsive, two constitutive-resistant, and two with acquired resistance. Only in constitutive-resistant tumors, PRA expression was silenced by DNA methylation. Next, we evaluated the effect of a demethylating agent, 5-aza-2'-deoxycytidine, on PRA expression and antiprogestin responsiveness. In constitutive-resistant tumors, 5-aza-2'-deoxycytidine treatment in vitro and in vivo restored PRA expression and antiprogestin RU-486 responsiveness. Furthermore, high levels of DNA methyltransferase (Dnmts) 1 and 3b were detected in these tumors. In conclusion, our results suggest that methyltransferase inhibitors in combination with antiprogestins may be effective in the treatment of constitutive-resistant carcinomas with a high DNA methyltransferase level.

R59022, a diacylglycerol kinase inhibitor, enhances the prostaglandin F2α (PGF2α)‐induced diacylglycerol (DAG) synthesis in Swiss 3T3 cells. It also potentiates the PGF2α‐mediated protein kinase C (PKC)‐dependent 80 kDa protein (80K) phosphorylation and initiation of DNA replication. R 59022 enhances the PGF2α mitogenic response by increasing the rate of entry into the S phase. Insulin does not cause 80K phosphorylation, and does not enhance its induction but it potentiates the PGF2α mitogenic response. These results suggest that mitogenically triggered fluctuations in DAG content and PKC activity play a pivotal role in controlling the PGF2α‐induced DNA synthesis while insulin acts via a different mechanism.

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)- or prostaglandin F2 alpha (PGF2 alpha)-induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2-80 microM concentration range, with both mitogens attaining 50% inhibition at 7.5 microM. LOV exerted its effect within 0-8 h following mitogenic induction. Mevanolactone (10-80 microM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF2 alpha response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF2 alpha-stimulated cells, LOV did not inhibit [3H]leucine or [3H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N' glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N' glycosylation process. These findings strongly suggest that either EGF or PGF2 alpha stimulations generate early cell cycle signals which induce mevalonate formation, N' glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed.

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F,, alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Elocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF& stimulation. In contrast tunicamicin dqes not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prerephcative phase is an important event controlling the mitogenic action of @GFr,.Swiss 3T3 cell; Prostaglandin F,; Tunicamycin; DNA synthesis; Lag phase N-Glycosylation involves several metabolic steps leading to the formation of the dolichol core oligosaccharide and its transference to the nascent polypeptide chains [15]. Tunisamycin (TM) inhibits the first step of the N-glycosylation process [16] and thereby alters many cellular processes [5-l 11. cultured Swiss mouse 3T3 cells have provided a useful model system to study the mechanisms that regulate proliferation [1'7]. These cells can be arrested in the GdG, phase of the cell cycle upon serum deprivation or when allowed to become coniluent [2,3]. Addition to such cultures of serum or a variety of growth factors, including prostaglandin F,, (PGF,,), stimulates the initiation of DWA synthesis after a constant lag phase of 14-15 h [2,3]. The rate of entry into S phase follows apparent first-order kinetics and can be quantified by a rate constant K [l&,19].Here we show that in confluent resting Swiss mouse 3T3 cells stimulated by PGF2, without or with insulin, TM inhibits the initiation of DNA synthesis, only if added within the first 8 h after mitogenic induction. Nevertheless, TRI does reduce the incorporation of ['4C]glucosamine and [31iJmannose into total cellular protein at any time after stimulation. These results sugpest that M-glycoprotein synthesis early during the lag phase plays an important role in the mitogenic response of PGF,,.2. MATERIALS AND METI-IODS 2.1. Cell culture, initiation of DNA synthesis assay and determination of rate constanl for entry into S phase Cell culture, conditions for the assay of DNA synthesis, labeling with [methyl-3H]thymidine and autoradiography were performed as previously described [ 191. The value of the rate constant K for entry into S phase was calculated as before [19]. Glucosamine, mannose and leucine incorporationCells were plated as for the assay of DNA synthesis [19]. For sugar labeling, the culture medium was removed and the cells were washed twice with 2.0 ml of serum-free medium minus glucose pre-warmed to 37% The cells then received 2.0 ml of the same medium containing the different stimuli and were labeled with 25 yM ['4C]glucosamine (I yCi/ml) and 50 ,KM ['PI] mannose (2.5 &i/ml) as indicated. For protein synthesis, stimulated cultures were exposed to [3H]leucine (2.5 ,uCi/ml). Thereafter, cells were processed and radioactivity was counted as described be...

Prostaglandin F2α (PGF2α), a mitogen for resting Swiss 3T3 cells, rapidly stimulates phosphorylation of an 80 kDa protein (80 K). 1‐Oleoyl‐2‐acetylglycerol (OAG) and 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) both protein kinase C (PKC) activators, also elicit 80 K phosphorylation. In contrast PGE1, PGE2 or PGF2β, which are non‐mitogenic in these cells, had tittle or no action on this event. However PGE1 and PGE2 potentiate the PGF2α proliferative effect but do not enhance its action on 80 K phosphorylation. These results suggest that PGF2α mitogenic induction involves PKC signalling pathway activation while its enhancement by PGE1 or PGE2 occurs through a different mechanism(s).