Molecular cloning of rat Krev-1 cDNA and analysis of the mRNA levels in normal and NMU-induced mammary carcinomas.

Human Krev-1 has been reported to reverse the transformed phenotype in a variety of cell types containing activated ras. Most mammary carcinomas induced by N-nitrosomethylurea in the rat have been reported to exhibit activated ras. Here we report that rat Krev-1 has a very high degree of homology to human Krev-1. Furthermore, Krev-1 expression is high in both normal and neoplastic mammary glands. No difference was found in expression levels in tumors with and without ras activation. These data argue against a tumor suppressor function for Krev-1 in the in vivo rat mammary gland.

Interaction with hyperthermia of tetrachloroplatinum(II)(Nile blue)2 and tetrachloroplatinum(II)(neutral red)2 in EMT6 murine cells and the murine FSaIIC fibrosarcoma.

Complexes of the tetrachoroplatinum(II) dianion with positively charged nuclear dyes were prepared in an effort to produce agents which gain ready access into the nucleus and become very cytotoxic at clinically relevant hyperthermia temperatures. Pt(Nile blue)2 and Pt(neutral red)2 are complexes of tetrachloroplatinum(II) with two closely related p-quinonediamine dyes. Pt(Nile blue)2 and Pt(neutral red)2 were only moderately cytotoxic to exponentially growing normally oxygenated or hypoxic EMT6 cells in vitro at pH 7.40 and 37 degrees C. At pH 7.40 and 42 degrees C and especially at 43 degrees C, however, Pt(Nile blue)2 became far more cytotoxic. At pH 6.45 Pt(Nile blue)2 became more toxic toward hypoxic cells (cell kill of 3.5 logs at 500 microM, 42 degrees C for 1 h). Pt(neutral red)2 became much more cytotoxic at pH 6.45 and 42 degrees C or 43 degrees C compared to pH 7.4, and the cell kill observed was similar in both euoxic and hypoxic cells (3 logs at pH 6.45, 43 degrees C with only 100 microM). Tumor cell survival studies in the FSaIIC murine fibrosarcoma demonstrated that both drugs killed in a dose-dependent log-linear manner. Hyperthermia treatment (43 degrees C, 30 min) immediately after either drug resulted in a dose modifying effect. The tumor growth delay produced by Pt(Nile blue)2 (100 mg/kg) was 4.6 days and by Pt(neutral red)2 (100 mg/kg) was 3.8 days. Both drugs were markedly improved by hyperthermia (tumor growth delay 1.4 days for hyperthermia; tumor growth delay 10.9 days for Pt(Nile blue)2 and 8.0 days for Pt(neutral red)2. Intracellular platinum levels were approximately 200 times higher after exposure of EMT6 cells to 25 microM of Pt(Nile blue)2 or Pt(neutral red)2 for 1 h at 37 degrees C than after exposure to the same concentration of cis-diamminedichloroplatinum(II). Treatment of cells with the drugs at 42 degrees C (1 h) resulted in no change in platinum levels with cis-diamminedichloroplatinum(II), but with Pt(Nile blue)2 and Pt(neutral red)2 an increase of 2- to 3-fold was found. Since previous work has shown that both of these complexes are active radiosensitizing agents, these new drugs seem quite well suited for further development as antitumor agents for use against solid tumors alone and in conjunction with hyperthermia and/or radiation therapy.

Effects of androgen withdrawal on the stem cell composition of the Shionogi carcinoma.

The parent Shionogi mouse mammary carcinoma is androgen dependent but cells that survive hormone withdrawal progress and give rise to an androgen-independent tumor. To determine whether renewed growth might be attributed to the persistence or partial recovery of an androgenic stimulus, we compared the amount of dihydrotestosterone and nuclear androgen receptor in parent and recurrent tumors. The whole tissue concentration of dihydrotestosterone in the parent tumor before castration was 1.40 +/- 0.46 (SE) as compared with 0.22 +/- 0.10 pmol/mg of DNA in the recurrent tumor. The initial concentration of nuclear androgen receptor in the parent was 0.65 +/- 0.12 pmol/mg of DNA; this was reduced to zero within 24 h after castration. Also in keeping with the androgen independence, no receptor was detected in the nuclear fraction of the recurrent carcinoma. In an attempt to relate malignant potential to nonhormonal factors associated with progression, we compared the proportions of androgen-dependent and -independent tumorigenic (stem) cells in parent and recurrent tumors using an in vivo limiting dilution assay. The difference observed, i.e., one stem cell per 4000 tumor cells in the parent versus one stem cell per 200 tumor cells in the recurrent carcinoma, was consistent with a marked enrichment of stem cells in the latter. The proportion of androgen-independent stem cells was also determined by assaying tumor takes in female hosts. The difference, i.e., one stem cell per 370,000 tumor cells in the parent versus one stem cell per 800 tumor cells in the recurrent carcinoma, demonstrated a striking 500-fold increase in androgen-independent stem cells resulting from androgen withdrawal. Unexpectedly, no enrichment of androgen-independent stem cells was evident in regressing parent tumors; rather, the proportion of such cells was very small, i.e., one androgen-independent stem cell per 2,200,000 regressing parent cells. This finding implies that the androgen-independent state of cells which survive androgen withdrawal may result from the ability of a small number of initially androgen-dependent stem cells to adapt to an altered hormone environment.

Changes in diacylglycerol and membrane associated protein kinase C activity reflect the growth status of xenografted human mammary carcinoma treated with 8-Cl-cAMP.

The intracellular accumulation of cAMP inhibits the growth of transformed cells in vitro and in vivo, and exposure to various cAMP analogs produces similar results. The influence of such analogs on the growth of neoplastic cells in vivo is less well defined, and the relevance of these analogs for the phosphoinositide pathway has not been established. The present report details the inhibition of tumor growth that occurred when human mammary xenografts were treated with 8-Cl-cAMP, the subsequent rebound in tumor growth that occurred when treatment ceased, and the levels of diacylglycerol and membrane-associated protein kinase C activity that characterized tumors in different growth states. Tumor levels of diacylglycerol and particulate PKC activity appeared to be influenced not only by treatment but also by treatment withdrawal. Changes in these entities tended to coincide with tumor growth rate, being relatively suppressed during growth stasis and markedly elevated during periods of rapid growth. The data presented do not establish a causal relationship. Thus, the concomitant changes noted in tumor growth and tumor levels of either diacylglycerol and membrane-associated protein kinase C may only be coincidental. Alternatively, they may indicate that cAMP analogs inhibit tumor growth in vivo by modulating the phosphoinositide pathway.

Modulation of mouse mammary tumor growth and linoleate enhanced metastasis by oleate.

This study examines whether oleate may influence the linoleate enhanced metastasis of line 4526 murine mammary tumors. In addition, the in vitro proliferative response of line 4526 to oleate and other selected fatty acids was assessed. Initially, the tumor cells were grown in a defined medium supplemented with palmitate, stearate, oleate, linoleate, linolenate or arachidonate. The unsaturated fatty acids stimulated and the saturated fatty acids inhibited proliferation compared to fatty acid-free medium. Next, we examined the effect of oleate on the linoleate enhanced metastasis of 4526 tumors by substituting oleate for saturated fat in isoenergetic diets containing high or low levels of linoleate. Oleate had no effect on metastasis in mice fed the high linoleate diets but it significantly increased metastasis in mice fed the low linoleate diets. Finally, the fatty acid compositions of tumors and mammary fat pads were compared to diet fatty acid compositions and metastatic frequency. Metastasis corresponded more closely to total unsaturated fatty acids than to total polyunsaturated fatty acids or to any individual fatty acid. These studies suggest that both mono- and polyunsaturated fatty acids may stimulate mammary tumor metastasis. However, the influence of dietary oleate probably depends on the level of linoleate and total unsaturated fatty acids in the diet.

Radioiodinated ligands for the estrogen receptor: tissue distribution of 17 alpha-[125I]iodovinylestradiol derivatives in normal and tumor-bearing adult female rats.

A series of three 125I-labeled 17 alpha-iodovinylestradiol derivatives previously demonstrated to have a selectivity for estrogen receptor tissues in immature female rats were selected for further evaluation in adult female rats. In normal adult female rats, the iodovinyl analogs of moxestrol (IV beta ME2), and moxestrol-3-O methyl ether (IV beta ME2-3-OMe) demonstrated high uterine uptake (0.296-0.437 and 0.135-0.199%ID-kg/g) and selectivity (10-18:1) over the 6 h time period. Subsequent evaluation in two tumor models indicated that [125I]V beta ME2 also possessed the highest tumor uptake and selectivity in the adult female rats bearing the estrogen responsive 9,10-dimethyl-1,2-benzanthracene(DMBA)-induced mammary tumors and that this was receptor mediated. An estrogen independent tumor, the transplanted Walker 256 mammary adenocarcinoma, showed no selectivity of radioligand uptake compared with nontarget tissues. The results suggest the applicability of this agent to the in vivo detection and characterization of estrogen responsive tumors in man.

Effects of OK-432 (Picibanil) on estrogen receptor levels and tamoxifen treatment in 7,12-dimethylbenz[a]anthracene-induced rat mammary cancers.

The effects of OK-432 (Picibanil) on estrogen receptor (ER) levels and subsequent tamoxifen (TAM) treatment were examined in 189 female Sprague-Dawley rats with 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancers. When OK-432 was administered (0.1 KE/kg i.p. once weekly) for 12 weeks to the rats after DMBA, the average ER level of the TAM-responsive tumors in the OK-432-treated group was significantly higher than that in the control group. The antitumor effect of TAM was significantly greater in the OK-432-treated group. When OK-432 was administered to rats with established DMBA tumors, the average ER levels did not change significantly after 2 or 4 weeks of treatment. ER levels in the control group (no treatment) fell significantly after 2 or 4 weeks. These results suggest that hormone dependence of DMBA-induced rat mammary cancers may be maintained or augmented by the administration of OK-432.

Effects of high dietary fat on the total DNA and receptor contents in rats with 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma.

The influence of high dietary fat on the malignant intensity and the hormone receptors of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in female Sprague-Dawley rats were analyzed by the tumor incidence and growth, the DNA histogram type, the DNA index, the S-phase fraction, and the estrogen receptor (ER) and progesterone receptor (PgR) assays. The rats were fed either a low-fat (0.5% corn oil) diet or a high-fat (20% corn oil) diet after the DMBA administration. Tumor incidences in the low-fat and the high-fat diet groups were 46 and 86%, respectively (p less than 0.01). Tumors in the high-fat diet group were also significantly larger than those in the low-fat group. Average tumor latent period was significantly shorter in the high-fat diet group, comparing with that in the low-fat diet group (p less than 0.01). Sixty-nine percent of the tumors in the high-fat diet group had aneuploid type, while only 8% of those in the low-fat diet group had aneuploid type. The DNA index and S-phase fraction also were significantly higher in the high-fat diet group (p less than 0.01). But the ER and PgR contents were not different between both groups. Therefore, these results suggest that a high dietary fat could increase the malignant intensity of the tumor but does not influence the hormonal responsiveness of these tumors.

Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues.

High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 micrograms whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplastic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor. Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland. Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.

Immunolocalization of collagenase in neoplastic epithelial cells.

The distribution of interstitial collagenase in a rat mammary carcinoma model system has been studied by immunocytochemistry. Rabbit antibodies were raised against collagenase from neoplastic epithelial cells which were derived from an anaplastic, invasive, rat mammary carcinoma (BC1). Specificity of the antibodies was determined by Western blot analysis which showed reactivity with the inactive procollagenase from conditioned culture medium of BC1 cells as well as with purified, active BC1 collagenase. Anti-BC1 collagenase antibodies did not recognize BC1 collagenase entrapped by the inhibitor, rat alpha-2-macroglobulin (alpha 2M), or collagenase derived from TPA-stimulated human fibroblasts. Anti-human fibroblast collagenase antibodies did not recognize BC1 collagenase, suggesting that the human-mesenchymal and rat-epithelial enzymes are immunologically distinct molecules. Collagenase was immunolocalized intracellularly in BC1 cells cultured in the presence of monensin. Neither BC1 collagenase, alpha 2M nor enzyme-inhibitor complexes were demonstrated in or around invading tumours by immunostaining of tissue sections of rat mammary carcinomas.

Receptors for D-Trp6-luteinizing hormone-releasing hormone, somatostatin, and insulin-like growth factor I in MXT mouse mammary carcinoma.

Membrane receptors for D-Trp6-luteinizing hormone-releasing hormone (D-Trp6-LH-RH), somatostatin-14 (SS-14), and insulin-like growth factor I (IGF-I) were estimated in MXT mammary cancers of mice using sensitive multipoint micromethods. The receptors were characterized in untreated animals and following in vivo treatment with microcapsules of the agonist D-Trp6-LH-RH and the somatostatin analog RC-160, which strongly inhibited tumor growth. In the control group, D-Trp6-LH-RH was bound to the single class of saturable, specific, noncooperative receptor sites (Kd, = 29.3 +/- 8.48 x 10(-9) M; Bmax = 4.55 +/- 0.31 pmol/mg membrane protein). Treatment with D-Trp6-LH-RH alone or in combination with RC-160 produced down-regulation of membrane receptors for D-Trp6-LH-RH on MXT mammary tumor cells. RC-160 alone and ovariectomy were without effect on D-Trp6-LH-RH receptors. On the membrane surface of MXT mammary cells, we found one class of high affinity, specific, saturable binding sites for SS-14 (Kd = 4.4 +/- 1.9 x 10(-9) M; Bmax = 0.58 +/- 0.21 pmol/mg membrane protein). Treatment with RC-160 alone or combined with D-Trp6-LH-RH significantly increased both the dissociation binding constant (Kd = 18.6 +/- 3.5 x 10(-9) and 10.1 +/- 0.7 x 10(-9) M, respectively) and the binding capacity (Bmax = 13.98 +/- 1.7 and 21.00 +/- 4.0 pmol/mg membrane protein, respectively). We also found specific binding sites (Kd = 3.01 +/- 0.15 x 10(-9) M; Bmax = 2.24 +/- 0.96 pmol/mg membrane protein) for IGF-I in the membrane fractions of MXT mammary cancers. Chronic treatment with D-Trp6-LH-RH and RC-160 alone or in combination, as well as ovariectomy, significantly decreased the dissociation binding constant of IGF-I membrane receptors on MXT mammary cells. Our results strongly suggest an important role of LH-RH, SS-14, and IGF-I in the growth of MXT mammary carcinoma. Changes in characteristics of receptors after treatment with analogs of LH-RH and SS-14 along with tumor growth inhibition provide additional support for the direct effect of these peptides on tumor cells. A possible significance of these findings as applied to a clinical environment is discussed.

Hormonal control of polyamine pools in experimental breast cancer in vivo: correlation with estrogen and progesterone receptor levels.

The present experiments were designed to test whether, in the hormone responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, polyamine levels are under hormonal control and whether they correlate with estrogen (ER) and progesterone (PgR) receptor content. We observed that tumor regression induced by ovariectomy was associated with a decline in putrescine (Pu), spermidine (Sd) and spermine (Sm). Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) to castrated rats restored tumor growth and contents of Pu and Sd to control values in a time dependent fashion while Sm levels were only modestly raised. The hormonal modulation of tumor polyamine levels was particularly obvious when the treatment effects on total pools (i.e. Pu + Sd + Sm) were analyzed. No significant correlation was observed between ER and PgR and polyamines in the tumors of intact rats as well as most of the treated groups. In contrast, a highly significant correlation was observed between ER and PgR levels. We conclude that in this experimental system cellular polyamine levels are hormonally regulated but are not correlated with the ER and PgR content of the tumor.

Effects of a high-linoleate and a high-alpha-linolenate diet on spontaneous mammary tumourigenesis in mice.

SHN mice were fed a high-linoleate diet, a high-alpha-linolenate diet or a control diet. Spontaneous mammary tumourigenesis was significantly inhibited in the high alpha-linolenate group compared to the other two groups, while little difference was observed among groups in the rates of lung metastasis. The dietary alpha-linolenate/linoleate balance affected the fatty acid patterns of tissue lipids. The triacylglycerol/phospholipid ratios and the fatty acid patterns were significantly different between the mammary glands and the mammary tumours. The results indicate that the dietary alpha-linolenate/linoleate balance affects the fatty acid composition and, in turn, spontaneous mammary tumourigenesis in mice.

Heterogeneous assembly of nascent core histones to form nucleosomal histone octamers in mouse FM3A cells.

We investigated the assembly of newly synthesized histones into nucleosomal histone octamers using an isopycnic centrifugation analysis of these proteins of mouse FM3A cells that had been labeled by culture with heavy amino acids. Cross-linked histone octamers or non-cross-linked core histones were separated electrophoretically from other proteins on sodium dodecyl sulfate/polyacrylamide gels before being banded to equilibrium in cesium formate/guanidinium chloride density gradients. The density of core histones rapidly came to equilibrium after culture of cells in the medium containing heavy amino acids for a time as short as 30 min, indicating that the density of histone octamers in the density-labeled cells reflects the content of these new (dense) core histones relative to old (light) ones in the octamer assembly. Cross-linked histone octamers from these cells were of heterogeneous densities as judged from a broad band in a density gradient. The position and width of the band was essentially unaffected, though the peak position of the density distribution within the band moved progressively from a less to a more dense area, when the time of culture was prolonged from 2 h to 16 h. It is concluded, therefore, that new and old histones are assembled into histone octamers in heterogeneous ratios in contradiction to a conservative or semi-conservative assembly model and it is also suggested that incorporation into chromatin of new histones is not necessarily restricted to newly replicated chromatin regions.

Effects of laser photodynamic therapy on tumor phosphate levels and pH assessed by 31P-NMR spectroscopy.

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.

Detection by in situ hybridization of messenger RNAs of collagen types I and IV in murine mammary cancer.

Ten breast carcinomas developing in C3H/Bi female mice were studied by an in situ hybridization technique using cDNA probes encoding alpha-I chains of collagens of types I and IV. The results obtained are compared to histochemical data on antibodies to collagens of types I and IV and ultrastructural observations on these tumors. Immunohistochemistry has revealed the presence of type-IV collagen in basement membranes lining intraductal and well-differentiated cancer nests. Type-I collagen was detected in the stroma surrounding these cells. There was no cellular labelling when these antisera were used. In situ hybridization has shown that type-IV collagen mRNA is detected in non-invasive intraductal and well-differentiated tumor cells and in endothelial cells in the stroma. Good correlation between detection of type-IV collagen lining these cells and evidence of mRNA by in situ hybridization was thus observed. Invasive cancer cells did not express hybridization grains with the type-IV collagen probe. Type-I collagen mRNA was visualized in stromal cells, probably fibroblasts and myofibroblasts as shown by electron microscopy. The most active cells were localized close to non-invasive areas. Our data indicate persistent in-vivo biosynthesis of type-IV collagen by some cancer cells that produce their own limitative environment; they suggest that type-IV collagen is not produced by invasive tumor cells and that stromal cells lining the non-invasive regions have a peculiar behavior.

Endocrine and antitumor effects of combined treatment with an antiprogestin and antiestrogen or luteinizing hormone-releasing hormone agonist in female rats bearing mammary tumors.

Rats bearing mammary tumors induced with dimethylbenzanthracene were treated with the antiprogestin mifepristone (RU486; 10 mg/kg.day, sc), the antiestrogen tamoxifen (400 micrograms/kg.day, sc), LHRH agonists administered by either sc injections (buserelin; 40 micrograms/kg.day) or implant (buserelin or zoladex), or combinations of mifepristone and tamoxifen or LHRH agonists. Single treatment with mifepristone or tamoxifen caused a significant inhibition of tumor growth (90% and 75%, respectively), but no tumor remission. In contrast, single treatment with LHRH agonists caused remission of mammary tumor growth by 50% (injection) or 70% (implant), respectively. Combined treatment with mifepristone and tamoxifen caused additive tumor growth inhibitory effects resulting in the same extent of tumor remission as that observed after treatment with LHRH agonist injections alone. Combination of mifepristone with either manner of LHRH agonist administration resulted in the highest tumor remission (approximately 75%). Significant reductions in cytosolic steroid (estrogen and progesterone) receptor contents of mammary tumors were noted after various treatment modalities. The most pronounced decrements were observed after combined treatment with mifepristone and tamoxifen (residual estrogen receptor; 10%; residual progesterone receptor, 0%). On the other hand, suppression of pituitary-ovarian function was most pronounced after treatment with LHRH agonist implants alone or in combination with mifepristone. It is concluded that combination treatment with an antiprogestin and an antiestrogen or an LHRH agonist may be of great value in the endocrine therapy of breast cancer.

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

Correlation of estrogen, progesterone, and androgen receptors in breast cancer.

Breast cancer was induced in female Holtzman rats by intragastric administration of 7,12-dimethylbenz[a]antracene (DMBA). At tumor maturity, biopsies of viable tissue were obtained, frozen, and then assayed for estrogen, progesterone, and androgen receptor content. By simple linear regression analysis, progesterone receptor levels significantly correlated with both estrogen and androgen receptor levels, whereas estrogen and androgen receptor levels did not correlate with each other. Multiple regression analyses further substantiated the predictive value of the progesterone receptor for the other two hormone receptors. Knowledge of breast tumor androgen receptor levels may further enhance the value of the estrogen receptor and progesterone receptor in hormonal responsiveness. Further, the progesterone receptor may be the most sensitive of the steroid hormone receptors for selecting patients likely to respond to hormonal therapy.

Platinum complexes with a selective action on estrogen receptor-positive mammary tumors.

Four (1,3-diaminopropane)dichloroplatinum(II) complexes, linked to 5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole by spacer groups of varying lengths, were synthesized and studied for their binding affinities for the calf uterine estrogen receptor. The RBA-values ranged from 1.0 to 4.4 (estradiol: RBA = 100). The endocrine activities of the complexes and their ligands, determined in the mouse uterine weight test, are low. All compounds entered comparative tests using estrogen receptor-positive and negative mammary tumors models. The receptor levels in these tumors were determined by a modified h.p.l.c. micro assay. In cell culture, a growth inhibiting effect was only observed in hormone-sensitive MCF-7 cells, but not in hormone-independent MDA-MB-231 cells. At 10(-6) molar, the cell number was generally decreased by 50%. In vivo, the growth of estrogen receptor-positive MXT mouse tumors was strongly inhibited whereas the hormone-independent MXT mammary tumors showed only a minor response. The most active compound was the platinum complex with a xylidene spacer group (4d-PtCl2) showing a reduction of tumor weight of 84% after 6 weeks of treatment (3 x 20 mg/kg/week). One of the complexes (4c-PtCl2) and its ligand were tested for activity against the hormone sensitive DMBA-induced rat mammary carcinoma. The inhibitory effect of the complex was close to that of cisplatinum. In these experiments, no sign of toxicity was observed. The selective effect on estrogen receptor-positive tumors make an endocrine mode of action both for the complexes and their ligands likely.