Progression to steroid insensitivity can occur irrespective of the presence of functional steroid receptors

A major problem in treatment of cancers arising in steroid-sensitive cells is their inevitable progression to a steroid-insensitive state; current therapies are based on the assumption that hormone insensitivity is associated with loss of receptor. We demonstrate for the first time that breast tumor cells can progress to steroid insensitivity in spite of functional steroid receptors. Transfection of the steroid-inducible LTR-C3 gene into unresponsive S115 mouse mammary tumor cells results in full inducibility of that gene with both androgen and glucocorticoid. Thus, although all known endogenous inducible parameters are lost, the steroid sensitivity of a transfected exogenous gene demonstrates that the machinery for steroid responsiveness is still fully functional. Furthermore, these transfected genes retain steroid sensitivity only while steroid is present; on prolonged withdrawal of steroid, they lose responsiveness, implying an epigenetic mechanism is involved.     

Expression of the androgen-dependent MMTV-specific orf gene in Shionogi 115 mouse mammary tumor cells

The Shionogi 115 (S115) mouse mammary tumor cells express the MMTV-specific 1.7 kb mRNA (orf) at a high level in the presence of androgens. In lymphoid cells the orf-gene encodes a superantigen which has an important role in establishing self-tolerance but in mammary and breast cancer cells the function of the orf gene is unclear. In the present work we studied the expression of the S115 mammary tumor cell orf sequence and its role in the androgen regulated growth of S115 cells. The cloning and sequencing of the cDNA specific for the 1.7 kb mRNA from the S115 mouse mammary tumor cells revealed a 990 bp DNA sequence with a 99.8% homology to the Mtv-17 proviral strain. There was a difference of only one amino acid (isoleu-tyr) in the coding region. A peptide was synthesized according to the hypervariable C-terminal part of the predicted protein and used to raise a rabbit antiserum. The anti-S115-orf antiserum immunoprecipitated an approximately 45 kDa protein from the metabolically labeled S115 cell lysates. In order to analyze the putative functions of the protein, the orf-sequence was linked to MoMLV-LTR and to the human ß-actin promoter in the mammalian expression vectors pLTRpoly and pHßAPr-1-neo, respectively, and transfected into NIH3T3 and S115 cells. NIH3T3 transfectants expressing orf mRNA did not show a transformed phenotype in vitro. The S115 orf transfectants proliferated somewhat more slowly than the vector transfected control cells in cell culture, both in the presence or absence of androgen, but there was no obvious change in the phenotype of S115 cells or in expression of the fibroblast growth factor 8 (FGF-8). This factor is activated by Mtv-6 integration and mediates androgen effects in these cells. Unexpectedly, however, the formation of tumors by S115 orf cells in nude mice was considerably prolonged and tumor growth retarded when compared with vector transfected control or parent S115 cells. The results suggest that MMTV-orf can be functional in breast cancer cells but the mechanism of the growth repressive effect in mammary tumor remains to be analyzed.     

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.     

Steroid hormone responsive cells in serum-free culture--analyses of hormone-mediated gene expression and cell growth

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.     

Antiandrogen ICI 176,334 does not prevent development of androgen insensitivity in S115 mouse mammary tumour cells

Many forms of endocrine therapy for steroid-sensitive tumours involve regimes of steroid agonist deprivation by administration of steroid antagonists. The partial or short-lived response to such therapy results from the inevitable progression of the tumour cells to a state of steroid insensitivity. Several cell culture systems have shown that steroid ablation results in loss of steroid sensitivity and we have used an in vitro model here to study the influence of steroid antagonists on this progression. Growth of androgen-responsive S115 mouse mammary tumour cells in the long-term absence of steroid results in a loss of androgen-sensitivity. We have studied here the effects of the pure antiandrogen ICI 176,334 on the growth of S115 cells and on their progression to steroid autonomy. Although a pure antiandrogen in its action on these cells with very low toxicity, it had no protective effect against loss of cellular or molecular androgen-responsive parameters. The clinical implications for endocrine therapy are discussed.     

Testosterone-induced responsiveness to androgen in Shionogi mouse carcinoma cells

Primary cell cultures from an androgen-dependent mouse mammary carcinoma, the Shionogi-SC 115 tumor, were cultured in the presence or absence of testosterone (50 nM). Characteristic changes in cellular morphology and cell growth were observed according to the presence or absence of the androgen. In testosterone-containing medium, cells formed individual clones, piling up one over another and showed no contact inhibition, whereas in the absence of the androgen, cells had a flattened morphology, they grew in a monolayer and cell multiplication was reduced. The testosterone-dependent changes were observed in culture as long as cells were maintained in androgen-containing medium. Only a few (3-5) days of culture in the absence of testosterone rendered cells irreversibly unresponsive to the androgen, and they could no longer produce tumours after inoculation in the host animal. Cellular proteins were analysed after culture in the presence or absence of testosterone. After [35S]methionine labelling of cells and SDS-PAGE of the cytosol, several proteins were specifically synthesized in the presence of testosterone, predominantly a 45 kD protein, which was not seen in the absence of the androgen. Conversely, a protein of 35 kD present in absence of the hormone disappeared in the presence of testosterone. The anti-androgen cyproterone acetate inhibited the characteristic cellular morphology, cell proliferation and protein synthesis observed in the presence of the the androgen. The antiprogestin and anti-glucocorticosteroid RU 486 also showed limited anti-androgen activity. The concentration of specific androgen receptor-binding sites did not change significantly after 3 months of culture with or without testosterone, i.e., in responsive and unresponsive cells.     

Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.     

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.     

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.     

Differential androgen sensitivity is associated with clonal heterogeneity in steroid metabolism, ornithine decarboxylase regulation and IL-1alpha action in mouse mammary tumor cells

Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.     

The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues.     

Approaches to studying the role of growth factors in the progression of breast tumours from the steroid sensitive to insensitive state

Progression from steroid sensitive to autonomous proliferation can be modelled in several cultured mammary tumour cell lines by long-term withdrawal of steroids. A feature of all the four systems studied thus far is that the basal growth in the absence of steroid increases with duration of steroid withdrawal until it reaches that obtained in the presence of steroid. It cannot be assumed that the increased proliferation in the absence of steroid is modulated by the same pathways as those stimulated by steroids in sensitive cells. Therefore, we feel that mechanisms of progression can best be studied via cell behaviour in the absence of steroid. With both the mouse S115 and human T-47-D systems, changes in sensitivity to several growth factors accompany progression; responses to TGF beta 1 are of particular interest in the T-47-D cells where this growth factor becomes stimulatory in the steroid insensitive state. This is accompanied by upregulation of TGF beta 1 mRNA. This upregulation of TGF beta agrees with the finding that ER - PR - primary human breast tumours contain more TGF beta 1 than do ER + PR + tumours; TGF alpha has the opposite pattern. Furthermore, only 40 and 30 kDa TGF beta species have been detected within cultured cells and primary tumours; TGF alpha exists in a 30 kDa form. The functions of these large forms of TGF alpha and TGF beta are unclear. Our conclusions from these experiments is that the increased proliferation in the absence of steroid accompanying progression may not be mediated by the same pathways as those perturbed by steroids in sensitive cells. Furthermore, TGF beta 1 may have different effects in steroid responsive and unresponsive cells.