Role of receptor occupancy in the transition from responsive to unresponsive states in cultured breast tumor cells

Progression from a steroid sensitive to insensitive state is characteristic of breast tumors, but little is known about the molecular mechanisms involved. Changes in steroid receptor can be associated with the progression. This paper reviews the cell culture data pertaining to loss of response and concludes that loss of receptor is a consequence rather than a cause of insensitivity. This view is based on evidence that loss of all response parameters occurs despite the presence of fully functional receptors as determined by transfection experiments. The postreceptor defect appears to be at the level of the hormone response element of the responsive genes and may involve DNA methylation. The implications of the model for human breast cancer biology are discussed.     

Morphological markers for the detection of introgression from cultivated into wild carrot (Daucus carota L.) reveal dominant domestication traits

Hybridisation and subsequent introgression have recently received much attention in the context of genetically modified crops. But crop–wild hybrid detection in the field can be difficult, as most domestication traits seem to be recessive, and the hybrid phenotype may also depend on the direction of the cross or environmental factors. Our aim was to develop a reliable set of morphological markers that differ between two wild and 13 cultivated carrots (Daucus carota L.) and to evaluate their inheritance in hybrid lines. We then examined these morphological markers in four F1 hybrids obtained by fertilising plants from the two wild accessions with pollen from two common carrot cultivars. Of the 16 traits that differed between the two carrot subspecies, three took intermediate values in the hybrids, eight resembled the cultivar parent (dominant domestication traits), two resembled the wild parent (domestication traits recessive), and three were not significant or growth condition‐dependent. Root:shoot ratio was seven times higher for cultivars than for wild plants, while still attaining equivalent total dry weight, which shows that dry matter production by the shoot is much higher in cultivars than in wild plants. High root:shoot ratios were also present in the hybrids. While we found no maternal effects, the type of cultivar used for pollination had an impact on hybrid characteristics. The morphological markers developed here provide insights into the mode of inheritance of ecologically relevant traits and can be useful for pre‐screening wild populations for hybrid detection prior to genetic analysis.     

Steroid regulation of transfected genes in mouse mammary tumour cells

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.     

Temperature-sensitive mutants for steroid-sensitive growth of S115 mouse mammary tumor cells

We have generated temperature-sensitive (ts) mutants for steroid-regulated anchorage-independent cell growth. Androgen-responsive S115+A mouse mammary tumor cells were mutagenized with ethyl methane sulfonate and the variants which were growth-arrested in suspension at the nonpermissive temperature of 41 degrees C were selected by killing dividing wild-type cells with the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine or cytosine arabinoside. Fifteen clones were isolated and characterized for morphology and growth properties. Three (ts21, ts27, ts33) of the phenotypic variants were ts for androgen-maintained anchorage-independent growth, two of them (ts27 and ts33) also for growth in monolayer. Growth arrest at 41 degrees C was not due to a defect in androgen receptor function in any of the mutant cell lines as shown by steroid binding assays and by the androgen-stimulated expression of both endogenous MMTV RNA and the transiently transfected LTR-CAT gene at the nonpermissive temperature. It remains to be determined for clone ts33 whether the defect is in postreceptor events of steroid action or in genes affecting general mechanisms of cell growth. However, since in clones ts21 and ts27 general cell growth remains functional at 41 degrees C under serum stimulation, defects may be in postreceptor steroid-related pathways. It is hoped that these mutants will provide a useful tool for study of steroid regulation of cell growth and in particular of the property of anchorage-independent growth.     

Amino acids attached to transfer ribonucleic acid in vivo.

1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results.     

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes.