Shionogi Mouse Mammary Carcinoma Research Articles

A Potential Role of Nuclear Matrix-associated Protein Kinase CK2 in Protection against Drug-induced Apoptosis in Cancer Cells

Protein kinase CK2 (CK2) has long been implicated in the regulation of cell growth and proliferation. Its activity is generally elevated in rapidly proliferating tissues, and nuclear matrix (NM) is an important subnuclear locale of its functional signaling. In the prostate, nuclear CK2 is rapidly lost commensurate with induction of receptor-mediated apoptosis after growth stimulus withdrawal. By contrast, chemical-induced apoptosis in prostate cancer and other cells (by etoposide and diethylstilbestrol) evokes an enhancement in CK2 associated with the NM that appears to be because of translocation of CK2 from the cytoplasmic to the nuclear compartment. This shuttling of CK2 to the NM may reflect a protective response to chemical-mediated apoptosis. Supporting evidence for this was obtained by employing cells that were transiently transfected with various expression plasmids of CK2 (thereby expressing additional CK2) prior to treatment with etoposide or diethylstilbestrol. Cells transfected with the CK2alpha or CK2alphabeta showed significant resistance to chemical-mediated apoptosis commensurate with the corresponding elevation in CK2 in the NM. Transfection with CK2beta did not demonstrate this effect. These results suggest, for the first time, that besides the commonly appreciated function of CK2 in cell growth, it may also have a role in protecting cells against apoptosis.

Antiandrogenic effect of crude extract of C-heavy oil

An oil spill accident happened to a Russian tanker Nakhodka with a cargo of heavy oil type C in the Sea of Japan on January, 1997 and the spilled oil severely polluted the coast of Japan and damaged the environment. In this study, androgenic and antiandrogenic activities of C-heavy oil crude extracts prepared with ethanol were evaluated on two androgen-responsive cell lines, a Shionogi mouse mammary carcinoma SC115 and a human prostate carcinoma LNCaP. Oils used in this study were the Nakhodka and a commercial C-heavy oils. Assessment of androgenic and antiandrogenic activities was made on the basis of effect on proliferation (SC115) and prostate specific antigen (PSA) production (LNCaP cells) in the absence and the presence of 0.5 nM dihydrotestosterone (DHT). While both extracts exerted almost no effect on the proliferation and PSA production of SC115 and LNCaP cells in the absence of DHT, the extracts significantly inhibited the DHT-induced proliferation and PSA production of the cells in the presence of DHT, indicating that the C-heavy oils contains antiandrogenic compounds. It has been known that androgen receptor (AR) expressed in LNCaP cells has mutation in ligand-binding domain and consequently, its transcription promoting action after ligand-binding is different from that of normal AR. Cyproterone acetate (CA), an androgen antagonist, and 17β-estradiol, an estrogen, stimulated PSA production and their stimulatory effects were additive to that of DHT in LNCaP cells, while CA inhibited DHT-induced proliferation and 17β-estradiol showed quite weak agonistic effect in SC115 cells. Therefore, a part of antiandrogenic effects of the oil extracts was considered to be mediated through mechanism other than direct transcriptional activation by activated AR. Then, a few polycyclic aromatic hydrocarbons (PAHs) that are considered not to bind to AR were examined for their androgenic and antiandrogenic effect. Benzo[ a]anthracene (BaA), benzo[ k]fluoranthene (BkF) and benzo[ a]pyrene (BaP) suppressed DHT-induced proliferation and PSA production of SC115 and LNCaP cells in a concentration-dependent manner. The antiandrogenic effect of the two heavy oil extracts was considered to be due in part to PAHs.

Effect of social housing condition on heat shock protein (HSP) expression in the Shionogi mouse mammary carcinoma (SC115)

Our previous studies have shown that social housing conditions can significantly alter the growth rate of the Shionogi mouse mammary carcinoma (SC115). The present study extended our investigations to the molecular level by examining stressor effects on the expression of a group of stress-responsive proteins, the heat shock proteins (HSPs). We hypothesized that HSP expression in SC115 cells may be altered by (a) different social housing conditions in vivo and (b) steroid hormone and growth factor exposure in vitro. Mice were reared in groups (G) or as individuals (I). Immediately following tumor cell injection, mice were rehoused from group to individual (GI), from individual to group (IG), or they remained in groups (GG). Tumor tissue was resected at 0.8 g or 3.0 g, as evidence suggests that tumor size affects HSP expression, which in turn affects proliferation. The data demonstrate that expression of HSP25, 70, and 90 was increased in tumors from mice in the IG compared to GG and GI mice, at both tumor weights examined. In addition, in IG mice, HSP90 expression was greater in 0.8 g compared to 3.0 g tumors. Under controlled culture conditions, hormones known to stimulate SC115 growth both in vivo and in vitro altered HSP expression. Physiological levels of dihydrotestosterone (DHT) and pharmacological levels of hydrocortisone (HC) upregulated expression of HSP25, whereas physiological levels of beta-estradiol (E2) upregulated expression of HSP90. These data are the first to demonstrate that a psychosocial stressor, a change in social housing condition, can induce differential HSP expression. Further, these data show that hormones that regulate SC115 tumor growth, also alter HSP expression.

Psychosocial stressors and mammary tumor growth: an animal model.

Stressful life events and the ability to cope with stress may play a role in the progression of breast cancer; however, the complex relationship between stressors and tumor growth is difficult to investigate in humans. Our studies have utilized the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice to investigate the effects of social housing condition on tumor growth rates and responses to chemotherapy. We demonstrate that, depending on social housing condition, mammary tumor growth and response to chemotherapy can both increase and decrease. We have examined the possible role(s) of 1) psychosocial variables, 2) testosterone and corticosterone, hormones altered by stress and known to stimulate SC115 cells in vivo and in vitro, 3) NK cells, one of the body's first lines of defense against tumor cells, 4) stress proteins, in mediating the differential tumor growth rates observed in our model. This review discusses the investigations we have undertaken to elucidate the mechanisms through which a psychosocial stressor, social housing condition, can alter tumor growth rate.

FGF8 over-expression in prostate cancer is associated with decreased patient survival and persists in androgen independent disease

Identification of prostate cancers at high risk of progression is difficult and a better understanding of how peptide growth factors influence cellular function might be useful. Fibroblast growth factors (FGFs) have been implicated in prostate cancer development. FGF8 was identified in the Shionogi mouse mammary carcinoma SC-3 cell line as an androgen-induced mitogen. We tested if FGF8 was over-expressed in human prostate cancer and if its expression correlated with clinical data and outcome. One hundred and six cases of prostate cancer and ten cases of BPH were examined. In situ hybridization was employed to detect FGF8 mRNA expression, which was identified within the malignant prostatic epithelium in 85/106 (80.2%) cases. Increased expression of FGF8 correlated significantly with higher Gleason scores (P=0.0004) and advanced tumour stage (P=0.0016). Using immunohistochemistry, we confirmed over-expression of the FGF8b isoform. Men with tumours which expressed high levels of FGF8 had worse survival (P=0.034), although FGF8 mRNA was not able to provide additional prognostic information in a multivariate analysis. Additionally, FGF8 expression was shown to persist in androgen independent prostate cancer. Using a range of normal adult tissues, FGF8 expression was restricted to neurones and the germinal epithelium in addition to the prostate. In vitro studies demonstrated that in the presence of neutralizing antibody to FGF8b there was significant inhibition of prostate cancer cell growth, confirming the biological significance of FGF8 in prostate carcinogenesis.

Interactive Effects of Psychosocial Stressors and Gender on Mouse Mammary Tumor Growth

We have previously demonstrated that social housing condition significantly affects the growth rate of the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice. The present study examined the effects of social housing condition and acute daily exposure to a novel environment on the growth rate of an androgen-independent variant of the AR SC115 carcinoma, designated SC115V, in male and female mice. Immediately following tumor cell injection, male and female mice that were reared as individuals (I) or in groups (G) of the same sex were rehoused either from individual to same-sex groups (IG) or from group to individual (GI), or remained in their group housing condition (GG). Approximately half the mice in each housing condition were subjected to acute daily exposure to novel environments (novelty stress), a treatment shown previously to increase the significant difference in tumor growth rates between male mice in the IG and GI housing conditions. The remaining mice were left undisturbed (no novelty stress). In the presence of acute daily novelty stress, the growth rate of the SC115V tumor was significantly increased in GI compared to IG males. However, no significant differences in SC115V tumor growth rates among nonstressed GI, IG, or GG males were observed. For females, in contrast to males, acute daily novelty stress significantly decreased tumor growth in GI compared to IG mice, whereas under nonstressed conditions, tumor growth rate was significantly increased in GI compared to IG females. Neither housing condition nor novelty stress altered estrous cyclicity, nor did the stage of the estrous cycle at the time of tumor cell injection influence tumor growth rates. These findings suggest that social housing condition and novelty stress may interact to produce differential effects on the growth rate of the SC115V tumor in male and female mice.

Effects of social housing condition and behavior on growth of the Shionogi mouse mammary carcinoma

We have demonstrated marked effects of social housing condition on the growth rate of the androgen-responsive Shionogi mouse mammary carcinoma. The present study investigated the possible role of psychosocial variables in modulating the differential tumor growth rates observed. Male DD/S mice were reared individually housed (I) or in groups (G) of three or five siblings or nonsiblings. Following tumor cell injection, mice either remained in their rearing conditions (II, GG) or were rehoused (IG, GI). Effects of group size, sibling relationship, dominance status, change vs. no change in housing condition, and direction of change (individual to group or group to individual) were examined. Home cage behaviors were monitored both prior to and following tumor cell injection and rehousing. Overall, mice in the GI conditions showed faster tumor growth rates than mice in the IG conditions. Mice in the II and GG conditions showed intermediate tumor growth rates. Differences in group size and sibling relationship prior to and following tumor cell injection and rehousing had no significant influence on tumor growth rates. However, both change in housing condition and direction of change following tumor cell injection/rehousing were significant variables in modulating differential tumor growth rates. Dominance status differentially influenced tumor growth depending on whether mice experienced a change in housing; in the IG conditions, dominant mice showed faster tumor growth whereas in the GG conditions, dominant mice showed slower tumor growth than subordinate mice. Increased fighting among mice in IG compared to mice in GG conditions may play a role in modulating differential tumor growth rates.

Synthesis and in vitro activity of 17 beta-(N-alkyl/arylformamido)- and 17 beta-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-3-oxo-5 alpha-androstan-3-ones as inhibitors of human 5 alpha-reductases and antagonists of the androgen receptor.

A number of 17 beta-(N-alkyl/arylformamido)- and 17 beta-[(N-alkyl/aryl)alkyl/arylamido]-3-oxo-4-aza-5 alpha-steroids were prepared from 17 beta-hydroxy-4-azasteroids and evaluated as inhibitors of human 5 alpha-reductase and antagonists of the androgen receptor. Jones' oxidation of 17 beta-hydroxy compounds gave the 17-keto-4-azasteroids, which were treated with amines and NaBH(OAc)3/NaBH3CN to give 17 beta-(N-alkyl/arylamino)-4-azasteroids 10-27. Alternatively, the above-indicated compounds were prepared from amines and 17-keto-4-azasteroids to form imines, which were then reduced with NaBH4. Formylation of amines 10-27 gave 17 beta-(N-alkylformamides) 28-41; however, acylation afforded 17 beta-[(N-alkyl/aryl)alkyl/arylamides] 42-53. In comparison to N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA; IC50 = 4.15 nM), 17 beta-(N-alkylformamido)-4-azasteroids were potent inhibitors of human type I 5 alpha-reductase, IC50 values of compounds 29, 30, 36, and 37 being measured as 3.05, 0.91, 2.19, and 2.35 nM, respectively. The structure-activity relationships suggest that the type I enzyme has preference for N-substituted straight alkyl side chains of four to five carbon atoms. On the other hand, formamides 32 (N-heptyl) and 33 (N-octyl), in addition to inhibiting the type I enzyme (IC50s = 9.57 and 16.9 nM, respectively), showed also strong inhibitory activity (IC50s = 14.0 and 18.4 nM, respectively) for human type II 5 alpha-reductase, in comparison to N-(1',1'-dimethylethyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (MK-906; IC50 = 4.53 nM). Other compounds in this series showed moderate activities (IC50 > 100 nM) on the type II enzyme. 17 beta-[(N-Alkyl/aryl)alkyl/arylamides] 45, 46, 48, and 51 exhibited highly potent inhibitory activity for human type I 5 alpha-reductase with IC50s of 1.77, 2.42, 2.93, and 5.44 nM, respectively, while moderate to no effect was observed on the type II enzyme (100 < IC50s < 1000 nM), except for compound 48 (IC50 = 3.75 nM). In another substitution pattern, N-aryl/alkylamides were studied; an electron-donating group increased the potency of compound 51, whereas an electron-withdrawing group decreased the potency of compounds 52 and 53 compared to parent compound 50. In addition to their 5 alpha-reductase activities, 17 beta-(N-alkylformamides) were also studied for their inhibitory activities on dihydrotestosterone (DHT)-stimulated proliferation of androgen-sensitive Shionogi mouse mammary carcinoma cells (clone SEM-107).(ABSTRACT TRUNCATED AT 400 WORDS)

Case Reports: Withdrawal Phenomenon With the Antiandrogen Casodex

No AccessJournal of UrologyCase Report1 Mar 1995Case Reports: Withdrawal Phenomenon With the Antiandrogen Casodex Peter T. Nieh, E. David Crawford, Howard I. Scher, and William K. Kelly Peter T. NiehPeter T. Nieh More articles by this author , E. David CrawfordE. David Crawford More articles by this author , Howard I. ScherHoward I. Scher More articles by this author , and William K. KellyWilliam K. Kelly More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(01)67644-4AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Total androgen blockade with castration and antiandrogens has become the primary therapy in metastatic prostate carcinoma. Several reports have been published on the flutamide withdrawal syndrome with a favorable response in patients with progression of disease after lengthy remission while taking combined hormone therapy. The experience with withdrawal of the experimental nonsteroidal antiandrogen casodex * in 3 patients is reported. All patients received casodex monotherapy initially, then luteinizing hormone-releasing hormone analogues were added on progression of disease (bone scan and prostate specific antigen (PSA)) and eventually casodex was withdrawn on further progression of disease (PSA and bone scan). References 1 : Prostate specific antigen decline after antiandrogen withdrawal: the flutamide withdrawal syndrome. J. Urol.1993; 149: 607. Google Scholar 2 : Response to flutamide withdrawal in advanced prostate cancer in progression under combination therapy. J. Urol.1993; 150: 908. Link, Google Scholar 3 : Flutamide withdrawal syndrome: its impact on clinical trials in hormone-refractory prostate cancer. J. Clin. Oncol.1993; 11: 1566. Google Scholar 4 : Use of the nonsteroidal anti-androgen Casodex in advanced prostatic carcinoma. Urol. Clin. N. Amer.1991; 18: 99. Google Scholar 5 : Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation. Biochemistry1992; 31: 2393. Google Scholar 6 : Aberrant response in vitro of hormone-responsive prostate cancer cells to antiandrogens. Prostate1989; 14: 103. Google Scholar 7 : Prostate-specific antigen decline after Casodex withdrawal: evidence for an antiandrogen withdrawal syndrome. Urology1994; 43: 408. Google Scholar 8 : Low androgen levels induce the development of androgen-hypersensitive cell clones in Shionogi mouse mammary carcinoma cells in culture. J. Natl. Cancer Inst.1988; 80: 1138. Google Scholar 9 : Tamoxifen withdrawal response: report of a case. Arch. Intern. Med.1989; 149: 449. Google Scholar 10 : Tamoxifen-induced tumor stimulation and withdrawal response. Cancer Treat. Rep.1979; 63: 1839. Google Scholar 11 : A controlled trial of leuprolide with and without flutamide in prostatic carcinoma. New Engl. J. Med.1989; 321: 419. Google Scholar From the Department of Urology, Lahey Clinic, Burlington, Massachusetts. Division of Urology, University of Colorado, Denver, Colorado. Genitourinary Oncology Service, Division of Solid Tumor Oncology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, Cornell University Medical College, New York, New York.© 1995 by American Urological Association, Inc.FiguresReferencesRelatedDetailsCited byKOJIMA S, SUZUKI H, AKAKURA K, SHIMBO M, ICHIKAWA T and ITO H (2018) Alternative Antiandrogens to Treat Prostate Cancer Relapse After Initial Hormone TherapyJournal of Urology, VOL. 171, NO. 2, (679-683), Online publication date: 1-Feb-2004.Schellhammer P, Venner P, Haas G, Small E, Nieh P, Seabaugh D, Patterson A, Klein E, Wajsman Z, Furr B, Chen Y and Kolvenbag G (2018) Prostate Specific Antigen Decreases After Withdrawal of Antiandrogen Therapy with Bicalutamide or Flutamide in Patients Receiving Combined Androgen BlockadeJournal of Urology, VOL. 157, NO. 5, (1731-1735), Online publication date: 1-May-1997. Volume 153Issue 3SMarch 1995Page: 1070-1073 Advertisement Copyright & Permissions© 1995 by American Urological Association, Inc.MetricsAuthor Information Peter T. Nieh More articles by this author E. David Crawford More articles by this author Howard I. Scher More articles by this author William K. Kelly More articles by this author Expand All Advertisement PDF downloadLoading ...

Endocrine mediation of psychosocial stressor effects on mouse mammary tumor growth

We have demonstrated that differential housing alters the growth rate of the androgenresponsive Shionogi mouse mammary carcinoma (SC115). In the present study we wished to determine if changes in plasma levels of hormones or a shift in the responsiveness of the tumor cells to hormones was responsible for the differential tumor growth rates observed. Plasma testosterone and corticosterone levels were assayed 24 h, 3 days and 1 week post tumor cell/vehicle injection. Also 3 weeks post injection androgen and glucocorticoid receptor binding capacity (B max ) and binding affinity (K d ) and the in vitro responsiveness of tumor cells to dihydrotestosterone and hydrocortisone were measured. At 24 h post injection, plasma testosterone levels were significantly increased in mice with large tumors, but remained low in mice with small tumors. Plasma corticosterone levels were significantly elevated in mice with small tumors compared to those of mice with large tumors at all time points measured. Androgen and glucocorticoid receptor binding capacity and binding affinity of tumor cells did not differ among groups. Further, all groups tested had the ability to respond to dihydrotestosterone and hydrocortisone in vitro. These data indicate that an effect of housing condition on plasma levels of steroid hormones may, in part, mediate the differential tumor growth rates observed in this model.

Androgen-responsive expression and mitogenic activity of schwannoma-derived growth factor on an androgen-dependent Shionogi mouse mammary carcinoma cell line

We report here, using the androgen-dependent SC2G cell line derived from Shionogi mouse mammary carcinoma SC115, that cDNA encoding mouse homologue of the rat schwannoma-derived growth factor (SDGF) was isolated and its protein product was identified to be an autocrine growth factor which is expressed in response to androgen in this hormone-dependent cell line. The androgen-independent SC1G cells derived from SC115 were shown to express SDGF even in the absence of androgen, while Northern analysis probed with mouse mammary tumor virus (MMTV) DNA showed that the androgen inducible transcriptional machinery remains functional in this hormone independent cell line.

Non-classical androgenic actions of RU38486 in androgen-responsive shionogi carcinoma 115 cells in serum-free culture

Antiglucocorticoid and antiprogestin RU38486 (RU486) stimulated the growth of highly androgen- and moderately glucocorticoid-sensitive SC-3 cells (a cloned cell line from Shionogi mouse mammary carcinoma 115) in a dose-dependent manner. A maximal 8-fold stimulation of growth by RU486 has been observed at 10 −7 M in a serum-free medium and its potency has been found to be almost the same as that of dexamethasone (Dex). The growth rate of SC-3 cells treated by triamcinolone acetonide (TA) or Dex combined with RU486 at 10 −9−10 −7 M was enhanced compared to cells treated by TA or Dex alone, indicating that RU486 had additive rather than antagonistic effects. Our previous study revealed that RU486 could compete with the specific uptake of [ 3H]testosterone in intact SC-3 cells at relatively low affinity and the present study showed that the stimulatory effect of RU486 on the growth of SC-3 cells was significantly inhibited by pure antiandrogen flutamine and that half-maximal inhibition by flutamine was achieved at 10 −6 M. Moreover, we demonstrated that the conditioned medium from RU486-stimulated SC-3 cells contained growth-promoting activity which caused a 3.5-fold increase in DNA synthesis by SC-3 cells in the absence of RU486 and which was abolished by treatment with heparin-Sepharose. These results indicate that RU486-induced growth of SC-3 cells may be expressed as an androgenic activity through androgen receptor and mediated by a heparin-binding growth factor.

Retinoic acid priming potentiates the induction of urokinase-type plasminogen activator by cyclic adenosine monophosphate in mouse mammary carcinoma cells.

Interactive regulation of gene expression by retinoic acid (RA) and adenosine monophosphate (cAMP) in mammary tumor cells was explored using Shionogi mouse mammary carcinoma cells (SC115) as a model and urokinase-type plasminogen activator (uPA) as a target gene product. Twenty-four hour treatment of SC115 cells with 100 nM RA, 1 mM 8-bromo-cAMP (BrcAMP), and 100 nM RA + 1 mM BrcAMP resulted in extracellular uPA activity increases of 1.4-fold, sevenfold, and 20-fold, respectively. These effects were dose-dependent with regard to both interacting members. Similar responses were obtained if 1 nM cholera toxin or 10 microM forskolin was used instead of the cAMP analog. Retinoids lacking the carboxylic acid function were inactive. The changes in uPA activity were accompanied by similar changes in uPA antigen concentration, as seen via Western blot analysis, and uPA mRNA abundance, as seen via Northern blot analysis. Actinomycin D, an inhibitor of RNA synthesis, blocked uPA stimulation by BrcAMP, suggesting that mRNA levels were transcriptionally regulated. The effect of BrcAMP on extracellular uPA activity was first evident at 2 h and peaked at approximately 6 h; the effect of RA alone and the synergistic response to joint treatment, however, followed a slower time course, requiring at least 12 h for initial expression and increasing gradually with time up to at least 48 h. Priming with RA for 48 h followed by extensive washing of the cells resulted in a threefold enhancement of the stimulatory effect of BrcAMP on uPA. Experiments utilizing the casein/plasminogen overlay method for the detection of uPA secretion by increased rate of uPA secretion per cell rather than to an increased fraction of uPA-secreting cells. Initial investigation of the mechanism of RA potentiation of cAMP responsiveness showed that RA did not alter cellular cAMP levels or total cAMP-dependent protein kinase A activity. Finally, the tumor promoter phorbol myristate acetate, an activator of protein kinase C, also increased SC115 cell uPA activity and synergized with RA. This raised the possibility that the enhancement of cAMP responsiveness by RA was indirectly mediated via an effect on protein kinase C. Experiments with protein kinase C-depleted cells, however, showed that this was not the case. In conclusion, RA treatment of SC115 cells potentiates the effect of cAMP on uPA expression at the single cell level via a partially irreversible mechanism independent of protein kinase C. The molecular target of RA and whether SC115 cell differentiation underlies the effect of RA remain to be established.

Loss of androgen dependence is associated with an increase in tumorigenic stem cells and resistance to cell-death genes

Complete remissions of the androgen-dependent Shionogi mouse mammary carcinoma are observed after androgen withdrawal but invariably the disease recurs and is refractory to further hormonal manipulations. To determine the proportions of androgen-dependent (AD) and -independent (AI) tumorigenic stem cells in parent and recurrent tumors and in vivo limiting dilution assay was developed. There was a marked enrichment of stem cells in the recurrent tumors ( 1 200 tumor cells) relative to the parent tumors ( 1 4000 tumor cells) when assayed in male hosts. By assaying tumor takes in female mice, the proportion of AI stem cells was found to be 1 370,000 tumor cells in the parent vs 1 800 tumor cells in the recurrent carcinoma; a 500-fold increase in AI stem cells resulting from androgen-withdrawal. Unexpectedly, no enrichment of AI stem cells was evident in regressing parent tumors; rather, the proportion of such cells was very small ( 1 2,200,000 tumor cells). This finding implies that the AI cells which survive androgen withdrawal may result from the ability of small number of initially AD stem cells to adapt to an altered hormonal environment. This adaptive process was further defined in terms of the disappearance of androgen receptors from the nucleus and the expression of androgen-repressed genes including the proto-oncogenes, c- fos and c- myc, and the cell death gene, TRPM-2; all of which are constitutively active in recurrent AI tumor cells. Overall, our results indicate: (1) the tumor mass consists mainly of differentiated cells; (2) stem cells initially are AD but at most the killing effect of androgen-withdrawal will be limited to 2–3 logarithms before compensatory adaptive mechanisms supervene; and (3) progression of stem cells to an AI state, in which they are resistant to the killing effects of cell death genes, might be prevented by the inhibition of androgen-repressed adaptive mechanisms which come into play when androgens are withdrawn.

Alterations in splenic natural killer cell activity induced by the Shionogi mouse mammary tumor

We have previously demonstrated that differential housing conditions alter the growth rate of the Shionogi mouse mammary carcinoma (SC115). The present study was undertaken to determine if natural killer (NK) cells are involved in mediating the effects of differential housing on SC115 tumour growth rate. Splenic NK cell activity was assayed at 24 h, 3 days and 1 week post-injection in both tumour- and vehicle-injected animals. Significant stimulation of splenic NK cell activity occurred 3 days post-injection of SC115 cells. However, no correlation was observed between the level of splenic NK cell activity and tumour growth rate induced by housing condition. We conclude that either splenic NK cell activity does not accurately reflect NK cell activity at the tumour site or that NK cells are not a significant regulator of the differential tumour growth rates seen in this model.

Effects of psychosocial stressors on mouse mammary tumor growth

We describe a sensitive and replicable animal model which demonstrates that psychosocial variables both increase and decrease tumor growth rate. The effects of social housing condition and daily exposure to novel environments were investigated in mice bearing the transplantable androgen-responsive Shionogi mouse mammary carcinoma (SC115). Being reared individually housed and remaining individually housed or being reared in a sibling group and then singly housed following tumor cell injection markedly increased tumor growth compared to that in mice remaining in their standard sibling rearing groups ( n = 2–3 per group), if animals were also exposed to acute daily novelty stress. In contrast, being reared individually and then moved to a larger social group ( n = 4–5 nonsiblings per group) markedly reduced tumor growth, both in the presence and absence of acute daily novelty stress.

Low androgen levels induce the development of androgen-hypersensitive cell clones in Shionogi mouse mammary carcinoma cells in culture.

The influence of the concentration of androgen present on the development of heterogeneous growth responsiveness to androgens was studied in an androgen-sensitive clone (SEM-1) of the Shionogi mammary carcinoma cell line incubated for up to 6 months in the presence of 0, 0.01, 0.3, or 100 nM dihydrotestosterone (DHT). In the absence of added androgen, there was a rapid increase in spontaneous cell growth, the increase being delayed by 2, 4, and 6 months when the cells were incubated with 0.01, 0.3, and 100 nM DHT, respectively. Conversely, the mitogenic effect of DHT decreased rapidly when originally androgen-sensitive cells were incubated with less than maximal concentrations of DHT. The most significant finding was the marked heterogeneity in the androgen sensitivity of the clones obtained after incubation for 2 months in the presence of less than maximal concentrations of DHT. In cells exposed to a maximal concentration of DHT (100 nM), only 2 of 22 clones were more sensitive to DHT (lower Km value) than the original clone, while 8 subclones were hyposensitive to DHT. On the other hand, when the cells were incubated for 2 months with a low (0.3 nM) concentration of DHT, 29% (7 of 24) of the clones were hypersensitive to DHT. Clones derived from cells treated with 0 or 0.1 nM DHT lost most of their responsiveness to DHT during the same time interval, with a simultaneous increase in spontaneous growth rate. The present data show that incubation of a clone of androgen-sensitive Shionogi carcinoma cells in the presence of a low DHT concentration, comparable to the circulating levels of DHT in castrated men, induces the development of androgen-hypersensitive cell clones that are able to grow on minute amounts of androgens. Such androgen-hypersensitive cells are likely to be unresponsive or resistant to antihormonal therapy. The present data emphasize the major importance of the hormonal environment on the maintenance, loss, and increase of tumor cell responsiveness to androgens.

Glucocorticoids stimulate the growth of mouse mammary carcinoma Shionogi cells in culture

The relative potency of a series of glucocorticoids to stimulate the growth of a cloned cell line (SEM-1) derived from the androgen-sensitive Shionogi mouse mammary carcinoma is proportional to their known affinity for the glucocorticoid receptor. The stimulatory action of glucocorticoids is not inhibited by the pure antiandrogen hydroxyflutamide while the antiglucocorticoids RU25593 and RU38486 cause 100% and 80% inhibitions of the activity of triamcinolone acetonide, respectively, thus indicating that the stimulatory effect of glucocorticoids on Shionogi cell growth is mediated by the glucocorticoid receptor. Such data indicate that not only androgens but also glucocorticoids should be taken into account when assessing the endocrine control of the growth of these mammary carcinoma cells.

Flow cytometric and karyotypic analyses of androgen-responsive and androgen-independent Shionogi mouse mammary tumours

The androgen-responsive (AR) Shionogi mouse mammary carcinoma is a heterogeneous tumour composed of AR and androgen-independent (AI) cells. We characterized the cells of the AR tumour and those of its AI derivative by flow cytometric analysis of DNA content and karyotypic analysis of metaphase spreads. Both tumours had diploid and near tetraploid populations of cells. However, the AR and AI malignant cells of these tumours both appeared to be polyploid. A decrease in the polyploid population of the AR tumour accompanied tumour regression following castration, but this population was restored when tumour growth resumed. Although karyotypic analysis of metaphase spreads showed wide variations in chromosome numbers among the polyploid population, the range, 55–88 chromosomes, was found in both AR and AI tumours. In addition, the same chromosome anomalies, including a marker chromosome, were identified in both tumours. Since the AR and AI malignant cells could not be distinguished on the basis of their DNA content or karyotype, the cell types may not represent genetically distinct populations of cells. The AR cells may undergo alterations in gene expression in adapting to their androgen-free environment.

Interaction of steroids with the nuclear envelope.

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90,000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.