Progesterone is an important regulator of normal and malignant breast epithelial cells. In addition to stimulating development of normal mammary epithelium, it can be used to treat hormone-dependent breast tumors. However, the mechanism of growth inhibition by progestins is poorly understood, and only a limited number of progesterone target genes are known so far. We therefore decided to clone such target genes by means of differential display polymerase chain reaction. In this paper, we describe an improved differential display strategy that eliminates false positives, along with the identification of nine positive (TSC-22, CD-9, Na ؉ /K ؉ -ATPase ␣1, desmoplakin, CD-59, FKBP51, and three unknown genes) and one negative progesterone target genes (annexin-VI) from the mammary carcinoma cell line T47D, which is growth-inhibited by progestins. None of these genes have been reported before to be progesterone targets. Regulation of desmoplakin, CD-9, CD-59, Na ؉ /K ؉ -ATPase ␣1, and annexin-VI by the progestin suggests that progesterone induces T47D cells to differentiate. Three of these genes were repressed by estradiol and up-regulated by the progestin. Estradiol treatment of T47D cells also leads to formation of lamellipodia and delocalization of two cell adhesion proteins, E-cadherin and ␣-catenin. All these effects were reversed by the progestin. These data suggest that estradiol dedifferentiates T47D cells, while progestins have the opposite effect. This may be linked to the capacity of progestins to inhibit tumor growth.

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A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.

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Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RAsensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-␣ expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid.Retinoids are a group of naturally occurring (e.g. all-transretinoic acid; RA 1 ) and synthetic analogs of vitamin A which play an important role in cellular growth and differentiation (1, 2). The actions of retinoids are mediated by two types of receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (3, 4). Both receptor types belong to the steroid-thyroid hormone receptor superfamily and regulate transcription in the presence of their ligands. RARs can be activated both by RA and 9-cis-RA, whereas only 9-cis-RA binds to RXRs (5, 6).Retinoids are highly effective in preventing chemically induced carcinogenesis in experimental animals (7) and can inhibit proliferation of a large variety of normal and neoplastic cell types in vitro (8). More recently the effectiveness of retinoids in the treatment and prevention of a number of human cancers has been established (9 -15).Unfortunately, lack of response to retinoid treatment and relapse of tumors are commonly observed. It is becoming increasingly clear that variations in metabolic rates of retinoids may be involved in the differences in retinoid response. Interindividual variation in the pharmacokinetics of retinoids has been reported for several malignancies, and a recent study suggested that high rate metabolism of RA is linked to an increased risk of squamous or large cell lung cancer (16 -18). In acute promyelocytic leukemia patients, relapse and resistance to RA have been associated with a rapid and marked decrease of retinoid levels in the plasma (19,20).Retinoid resistance has also been docu...