Effect of exogenous epidermal-like growth factors on mammary gland development and differentiation in the estrogen receptor-alpha knockout (ERKO) mouse

The development of the mouse mammary gland requires the interaction between several different ovarian and pituitary hormones such as estrogen, progesterone and prolactin as well as several locally-derived growth factors in the mammary gland such as epidermal growth factor (EGF), transforming growth factor (TGF), amphiregulin (AR) and heregulin (HRG). The focus of this study was to investigate the degree of mammary growth and differentiation in the adult, virgin mammary gland of wild type (wt) and estrogen receptor knockout (ERKO) females that lack estrogen receptor (ER) after reciprocal transplantation into the cleared mammary fat pad of virgin wt or ERKO mice. In addition, we assessed the local response of ERKO mammary tissue to TGF or HRG1 delivered from slow release-Elvax pellets. Our initial results indicated that when we transplanted virgin wt mammary tissue into ERKO mammary fat pads, mammary morphogenesis failed to occur. However, when transplanted virgin ERKO mammary tissue was transplanted into fat pads of virgin or pregnant wt mice, the development and differentiation of lobuloalveoli was readily observed. In addition, treatment of the virgin ERKO mammary gland with TGF or HRG1 stimulated ducts to undergo localized branching and growth and both growth factors induced secretory differentiation as evidenced by the production of milk proteins, caseins and/or whey acidic protein (WAP). The results from this study imply that in ERKO mammary tissue, ERKO ductal epithelium has the capacity to proliferate and differentiate in response to non-estrogenic, morphogenic stimuli.     

Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague–Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r=0.05, p>0.80), whereas there was a significant correlation in ductal epithelium (r=0.86, p=0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p<0.03). All carcinomas from vorozole-treated rats expressed PR (2.5–60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r=0.42, p=0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

Immunological detection and quantitation of alpha transforming growth factors in human breast carcinoma cells

Summary Alpha transforming growth factors (TGFs) were immunologically detected in the concentrated conditioned medium (CM) prepared from four human breast cancer cell lines and from primary cultures of human mammary epithelial cells, and in the tissue extracts prepared from normal, benign, and malignant breast biopsies. Immunoreactive TGFs were quantitated by a competitive radioimmunoassay (RIA) using affinity-purified polyclonal sheep anti-rat TGF antibodies which react with human TGF but not with human epidermal growth factor (EGF). The relative level of RIA-detectable TGFs in the CM from the breast cancer cell lines MCF-7, ZR-75-1, T47-D, and MDA-MB-231, and from the CM of primary cultures of human mammary epithelial cells, ranged from 0.02 to 0.85 ng/ml. MCF-7 or ZR-75-1 cells grown in the presence of 17-estradiol (10^–8 M) for 48 h were found to release two- to three-fold more TGFs into their CM than the same cells grown in the absence of estrogen. In detergent extracts prepared from normal breast tissue, a benign fibrocystic lesion, fibroadenomas and primary breast carcinomas, the relative TGF concentrations were found to range from 1.5 to 6 ng/mg cell protein. No significant correlations were found between the TGF levels and the pathological state of the tissues, the estrogen receptor status of the tumors, or the relative amounts of theras gene protein p21ras in the tissues as determined by Western immunoblot analysis. The question of biological relevancy of TGF for human mammary tumors will require further studies on (a) synthesis and turnover of TGF, (b) the relationship between immunoreactivity and biological activity of TGF, and (c) differences in biological responsiveness of mammary tumor cells.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations

Summary The antiproliferative effects of human interferons (IFNs) and double-stranded RNAs (dsRNAs) were studied in five human glioma cell lines. Dose response curves were generated over a 72 hour treatment period. The concentration of interferon or double-stranded RNA necessary to produce a 50% antiproliferative response (GI₅₀) was calculated by linear regression analysis. Two cell lines were more sensitive to IFN- than to IFN-, one cell line was more sensitive to IFN- than to IFN- and two cell lines had approximately equal sensitivities to both interferons. All cell lines showed some sensitivity to either IFN- or IFN-. IFN- had no antiproliferative effect on any of the cell lines. In addition, only one of the cell lines displayed sensitivity to dsRNA, in which the response to poly(I) · poly(C) was greater than that to a mismatched analogue of poly(I) · poly(C), r(I)n · r(C₁₂,U)_n (Ampligen). There was no correlation between the sensitivities to type I IFNs ( and ), type II IFN () or the dsRNAs. The antiproliferative effect of combinations of IFNs, or IFNs and Ampligen, was studied in one of the cell lines. A significant synergistic antitumor effect was seen with all of the IFN/Ampligen combinations (p < 0.02), including IFN-/Ampligen, even though these cells were resistant to IFN- alone. Synergy was also seen in the IFN-/IFN- (p < 0.02) and IFN-/IFN- (p < 0.05) combinations. The IFN-/IFN- combination gave an additive antitumor effect. These results indicate that IFN- and IFN- alone or combinations of type I IFNs, type II IFNs and Ampligen can be effective in inhibiting the growth of glioma cells.     

Pharmacokinetic and pharmacodynamic comparison of fluoropyrimidine derivatives, capecitabine and 5′-deoxy-5-fluorouridine (5′-DFUR)

Purpose Capecitabine is a three-step prodrug that was rationally designed to be a more effective and safer alternative to its intermediate metabolite, 5-deoxy-5-fluorouridine (5-DFUR). We compared the pharmacokinetics/pharmacodynamics of these drugs in metastatic breast cancer patients.Methods Six patients received oral capecitabine at 1657 mg/m² twice daily and 17 received 5-DFUR at 400 mg three times daily. Both drugs were administered for 21 days followed by a 7-day rest.Results Median daily 5-DFUR AUC was significantly higher for capecitabine than for 5-DFUR (81.1 vs 32.6 mmol h/l; P=0.01). Following treatment with 5-DFUR, the median AUC and C_max of 5-DFUR tended to be higher in patients with a partial response (3.83 g h/ml and 4.88 g/ml) and stable disease (6.46 g h/ml and 4.96 g/ml) than in those with disease progression (2.53 g h/ml and 1.36 g/ml). The AUC and C_max of 5-DFUR was significantly related to overall survival.Conclusions These results support the superiority of capecitabine over 5-DFUR.     

The effect of flutamide and tamoxifen on sex hormone-induced mammary carcinogenesis and pituitary adenoma

We have established a female Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis. Based on the previous finding that the dose of testosterone affects only the latency period of mammary cancer, not the final incidence and that androgen upregulates apoptotic activity in pre-malignant mammary glands, we hypothesised that estrogen is the initiator and androgen the promoter for hormonal mammary carcinogenesis of the rats. In the present study, rats were treated with the sex hormones together with flutamide and tamoxifen for both short term (7 and 13weeks) and long term (12months) durations. We showed that tamoxifen could totally inhibit mammary carcinogenesis while flutamide cause a delay and reduction in tumour incidence in the 12months treatment term. Blocking effect of flutamide and tamoxifen on T+E₂ (testosterone and 17-estradiol) short-terms treatment was demonstrated by the similar histological changes identified in the mammary glands of the T+E₂ and drug treated rats to that of the age matched E₂ and T controls, respectively. These findings give further support for the role of estrogen and androgen in mammary carcinogenesis. Autopsy of the tumour bearing rats showed presence of pituitary macroadenoma causing compression and atrophy of the brain stem. Immunohistochemical staining of these adenomas showed a predominance of prolactin-secreting cells. Serum assay also showed a corre-sponding increase in circulatory prolactin level. Tamoxifen was also effective in blocking the formation of pituitary adenoma in the sex hormone treated rats. Pituitary size and level of prolactin were higher in the T+E₂ +flutamide group than the T+E₂ group in both short-term and long-term treatments. It suggests that testosterone may have a role in counteracting estradiol stimulation on the pituitary lactotropes although it is synergistic to estrogen in mammary carcinogenesis. Pituitary adenomas were found in all rats that developed mammary adenocarcinoma but not vice versa suggesting that prolactin level elevation alone cannot lead to mammary tumorigenesis. The animal model, in addition to mammary carcinogenesis, may be useful for investigation of anti-estrogen therapy in pituitary adenomas.     

A phase II study to evaluate the combination of fludarabine, mitoxantrone and dexamethasone (FMD) in patients with follicular lymphoma

Background:Molecular response is being investigated as atherapeutic goal in follicular lymphoma (FL). High response rates in FL withthe fludarabine combination FMD have been associated with molecularremission. A phase II study of FMD in FL was therefore conducted. Patients and methods:Fifty-four patients, ten of whom were newlydiagnosed received FMD. Forty-four percent of the previously treated patientshad chemoresistant disease. Treatment comprised: fludarabine 25mg/m² days 1–3, mitoxantrone 10 mg/m² day 1, anddexamethasone 20 mg days 1–5. Blood/bone marrow was collected forquantitation of t(14;18) by real-time PCR. Results:The overall response rate was 37 of 54 (69%),complete responses being seen in 11 patients (20%), with no differencebetween newly diagnosed and the previously treated patients. However, theresponse rate in chemosensitive relapse was 84%, compared to44% in patients in whom the last prior regimen had failed. Molecularresponses were seen in 17 of 25 and PCR negativity in 8 of 25, althoughmolecular and clinical responses did not always correlate. Toxicity wasmoderate, 19 patients required admission. However, in 6 of 12 patients,subsequent G-CSF mobilised stem cell harvests failed. Conclusions:FMD was well tolerated but with a lower than expectedresponse rate. Molecular responses were seen in the majority of respondingpatients however, molecular remission was rare.     

Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts

Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM-1, and integrin chains 2, 3, 4, 5, 6, v, 1, 3 and 7 failed to inhibit breast cancer cell migration through bone marrow fibroblasts. Inhibitors of matrix metalloproteases, 1, 10-phenanthroline, Ro-9790, TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers suggesting a role for these enzymes in the migration of breast cancer cells through bone marrow adherent layers. Co-culture of MDA-MB-231 cells and bone marrow fibroblasts resulted in augmentation of the levels of the matrix metalloproteases MMP-1 and MMP-2 in culture supernatants. Soluble factors produced by bone marrow fibroblasts were responsible for the increase in MMP-1 levels. However, maximal MMP-2 production was dependent on direct contract between the breast cancer cells and the bone marrow fibroblasts. Modulation of MMP production by cell–cell contact or soluble factors suggests a mechanism by which breast cancer cells can enhance their ability to invade the bone marrow microenvironment.     

Intermittent Blood Flow in Solid Tumours – an under-appreciated Source of ‘drug Resistance’

As the search for improved anti-cancer drugs continues, new paradigms concerning the reasons for clinical failures in common human solid tumours are also evolving. Classical drug resistance is now perhaps less often invoked to explain lack of treatment efficacy than are newer concepts, including contact resistance, tumour heterogeneity, regrowth resistance, and physiological barriers to drug delivery. This commentary will explore the resistance of solid tumours to chemotherapy from yet another, largely ignored perspective: that of tumour-specific fluctuations in blood flow. Transient decreases in blood flow have significant implications for delivery of chemotherapeutic agents, cellular responsiveness to those agents, and the regrowth potential of the surviving tumour cells.     

On the development of an interstitial radiation protocol for a multicenter consortium. Experience with permanent low-dose rate and temporary high-dose rate125I implants in ‘failed’ and ‘newly diagnosed’ glioblastoma patients: Quality assurance methodology and a possible future adjuvant for therapeutic enhancement

Summary Three interstitial implant trial groups (one permanent low-dose rate¹²⁵I and two temporary high-dose rate¹²⁵I implants) in glioblastoma patients (newly diagnosed and failed) were compared to non-randomized similar control groups for efficacy. The results formed the basis for the BTCG 87-01 national implant trial. The pilot trial demonstrated: 1) the effectiveness of a temporary high-dose rate¹²⁵I implant in failed and newly diagnosed patients; 2) the ability of a multicenter consortium to adhere to a standard protocol; 3) a methodology to insure quality assurance; and 4) the possibility of the future adjuvant application of hyperthermia using a single catheter system.     

Selective inhibition of cell proliferation and DNA synthesis by the polysulphated carbohydrate ι-carrageenan

-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of -carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the antiproliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of -carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC₅₀ value), approx. 0.5 g/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of -carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by -carrageenan (IC₅₀ value, 5.5 g/ml) and the cells were arrested at the G1/S boundary. -Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor (TGF) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody IR3 enhanced the inhibitory activity of -carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by -carrageenan. -Carrageenan had low anti-coagulant activity. -Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.     

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

Summary In order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent -adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize -adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthraene (DMBA) administration in the rat. The binding of [¹²⁵I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [¹²⁵I]CYP binds to a single class of high affinity sites (114 ± 2.1 fmoles/mg protein) at an apparent K_D value of 38.0 ± 0.3 pM. The order of potency of a series of agonists to compete for [¹²⁵I]CYP binding is consistent with interaction with a ₂-subtype receptor: zinterol > (–)isoproterenol > (–)epinephrine» (–)norepinephrine. In addition, the potency of a series of specific ₁, and ₂ synthetic compounds to displace [¹²⁵I]CYP in mammary tumors is similar to their potency in typical ₂-adrenergic tissues. The binding of [¹²⁵I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (–)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [¹²⁵I]CYP performed on frozen sections revealed the presence of specific -adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18–22 months) female rats have high levels of -adrenergic receptors. Castration decreased the concentration of [¹²⁵I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and -adrenergic receptor concentration. The presence of -adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.     

Metachronous seeding of lymph node metastases in rats bearing the MT-100-TC mammary carcinoma: The effect of elective lymph node dissection

Summary Following the introduction of cancer cells into the lymphatic system, metastases in down-stream lymph nodes often appear in a sequential manner. This could be due to synchronous seeding of the in-line nodes with progressively diminishing numbers of tumorigenic cancer cells, or alternatively, by discrete, stepwise (metachronous) seeding of down-stream nodes by up-stream nodal metastases acting as generalizing sites. Metachronous seeding to local lymph nodes is potentially curable by elective lymph node dissection; synchronous seeding is not.Synchronous versus metachronous seeding of lymph node metastases was investigated using the MT-100-TC mammary carcinoma injected into the hind foot-webs of rats. When the primary tumor was removed by amputation one week after injection, 1/15 animals survived; in contrast, removal of the draining popliteal lymph node in addition to the primary lesion, resulted in 8/19 long-term survivors. At this time, occult metastases detectable by bioassay butnot by conventional histology, were present in all draining popliteal nodes and in 60 percent of lungs. The fact that some amputees were cured when the popliteal node was removed, indicated the metachronous nature of nodal metastases in this system. Further, recurrence of nodal and lung metastases in those amputees in which the popliteal node was left intact, identified the popliteal node as a generalizing site. By the time popliteal node involvement was evident by conventional histology, micrometastases were present in down-stream nodes, and accordingly, removal of the popliteal node and the primary lesion at this time was not curative.     

Biological and clinical evaluation of Lanreotide (BIM 23014), a somatostatin analogue,in the treatment of advanced breast cancer

Summary Recently, compounds having pure antiestrogenic activity have become available. In this study, we examined the activity of the new steroidal antiestrogen EM-170 (N-n-butyl, N-methyl-11-(16-chloro-3,17-dihydroxy-estra-1,3,5-(10)-trien-7-yl) undecanamide) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma stimulated by treatment with estrone (E₁), a steroid known to play an important role as precursor of 17-estradiol (E₂), especially in postmenopausal women. Twenty-five days after ovariectomy (OVX), tumor volume in control OVX animals decreased to 51.4 ± 11% of the initial volume; treatment with E₁, administered by Silastic implants, stimulated tumor growth to 179 ± 21%. Treatment with the antiestrogen EM-170 at a dose of 200 µg (twice daily) not only completely reversed the stimulatory effect of E₁, but also inhibited tumor growth to 30.5 ± 9.6%, an effect that is 41% (P < 0.01 vs OVX control) greater than that of ovariectomy alone. At a relatively low dose of 40 µg (twice daily), 20 days of treatment with EM-170 reversed by 55% the stimulatory effect of E₁ (1.0 µg, subcutaneously, twice daily) on tumor growth in OVX animals. On the other hand, the antiestrogen also induced a significant inhibitory effect on 17-hydroxysteroid dehydrogenase (17-HSD) activity in the DMBA-induced mammary tumors, an effect that is in agreement with the marked reduction caused by the same treatment on tumor estradiol (E₂) levels in E₁-treated OVX animals. The present data show that the new steroidal antiestrogen EM-170 exerts a potent inhibitory effectin vivo on E₁-stimulated growth of DMBA-induced mammary tumors, an effect that is probably mediated by both its antiestrogenic activity at the receptor level and its inhibitory effect on 17-HSD, thus inhibiting local E₂ formation and facilitating the action of the antiestrogen at the receptor level.     

Mitotic rate,DNA distribution,and chromatinin situ sensitivity to heparin in breast cancer

Summary The purpose of this study was to characterize breast carcinomas by cell kinetic parameters. Mitotic rate (MR) and flow cytometrically (FCM) measured cell cycle distribution as well as chromatin testing in situ employing heparin for determination of activated chromatin, provided the following results:MR counted in 73 unselected carcinomas showed an increase up to a tumor size of 4.2cm (p < 0.05); beyond this diameter, the MR was found to decrease.In T1-T2 carcinomas, cell cycle stage analysis yielded higher percentages of cells in S and G₂M phase for ductal (13% and 12%, N = 22) than for lobular (8% and 7%, N = 8) node-negative carcinomas (p < 0.002). In ductal carcinomas, lymph node involvement was reflected by higher % G₂M values (15%, N = 26) compared with negative cases (12%, N = 22) (p < 0.05).Ductal node-positive T3-T4 carcinomas (N = 10) revealed a higher % S value (16%) than their T1-T2 counterparts. A correlation between MR and % G₂M was established only up to a tumor size of 4.2 cm (r = 0.39, p < 0.05).A highly sensitive (H) and a poorly sensitive (P) subgroup of carcinomas with respect to heparininduced changes in fluorescence intensity of the G_1/0 peak of the DNA aneuploid cell line were identified, as previously shown [1]. These subgroups were here updated with a larger number of carcinomas and were limited to T1-T2 cancers (N = 57). Group H included more younger patients (p < 0.005), less cases with nodal involvement in ductal carcinomas (p < 0.05), and lower % G₂M values in lobular node-negative cases (p < 0.05), than group P. DNA diploid cells always existing in DNA aneuploid carcinomas are more sensitive than their aneuploid counterparts (p < 0.01); however, they strengthen the stratification to H and P. We suggest H carcinomas to be less aggressive than P carcinomas. Small breast carcinomas are recommended to cell kinetic investigations for individualizing adjuvant therapy.     

In vitro biotransformation of estradiol by explant cultures of murine mammary tissues

Summary In vivo experiments have demonstrated a correlation between the extent of 16-hydroxylation of estradiol and incidence of mammary cancer. The ability of mammary ductal epithelium (MDE), the site for neoplastic transformation, to metabolize estradiol or to accumulate estradiol metabolites has not been unequivocally established. Using a newly developed mammary explant culture system and a radiometric assay, we have compared the site-specific metabolism of estradiol (E₂) by the C-17-oxidation and C-16-hydroxylation pathways in mouse tissues that differ in relative risk for mammary cancer. A comparison between MDE (target tissue) and liver (nontarget tissue) from NFS (low risk) and C3H/ouj (high risk) mice revealed that: a) increase in C-17-oxidation was similar in MDE and liver from the two strains, and b) while C-16-hydroxylation was similar in liver from the two strains (p = 0.5, n.s.), it was increased 4-fold in the MDE from the high risk C3H/ouj strain relative to that from the low risk NFS strain (p = 0.001). Furthermore,in vivo administration of progesterone resulted in modulation of cell proliferation as well as of E2 metabolism in mammary explant cultures. The effect of progesterone depended upon the presence of the MtV-2 proviral gene. This study demonstrates that mammary explants can extrahepatically metabolize estradiol. The specific risk-related increase in C-16-hydroxylation suggests that intrinsic metabolic ability of the target tissue leading to the formation of 16-hydroxyestrone from estradiol may be a determinant in the relative risk for developing mammary cancer.Abbreviations MMTV Mouse Mammary Tumor Virus - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 16-OHE₁ 16-hydroxyestrone - MDE Mammary Ductal Epithelium - HN Hyperplastic Nodule - MtV-2 Proviral Gene of Mammary Tumor Virus - GRS^MtV-2+ GRS strain of mouse carrying MtV-2 proviral gene - GRS^MtV-2- GRS strain of mouse lacking MtV-2 proviral gene - 020^MtV-2- 020 strain of mouse lacking MtV-2 proviral gene - 020^MtV-2+ 020 strain of mouse carrying MtV-2 proviral gene     

Role of Ceramide During Cisplatin-induced Apoptosis in C6 Glioma Cells

Cisplatin is commonly used for the treatment of malignant brain tumors. However, the mechanisms of cell death by cisplatin are not fully understood. Therefore, the present study was designed to elucidate the apoptotic signaling pathway(s) activated by cisplatin in a C6 rat glioma cell line. C6 cells were treated with various concentrations of cisplatin (0.2–10g/ml) for 24–72h. At 10g/ml cisplatin, over 90% of the cells became dead at 72h. Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering. Even in cells treated with 1.5g/ml cisplatin, typical apoptotic cells were observed at 72h. The intracellular level of ceramide, measured Escherichia coli diacylglycerol kinase markedly increased during 24–72h after the addition of 10g/ml cisplatin. The activity of caspase-3(-like) proteases increased and reached a peak at 48h. Inhibitors of caspases reduced the number of apoptotic cells. Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formation, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin. In contrast, pretreatment of the cells with N-oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis induced by cisplatin. Furthermore, OE enhanced sensitivity of the cisplatin-resistant cells to cisplatin. These results suggest that ceramide is closely implicated in apoptosis of glioma cells by cisplatin through activation of caspase-3(-like) proteases.     

Cytokine‐regulated urokinase‐type‐plasminogen‐activator (uPA) production by human breast fibroblasts in vitro

It has been shown that, in breast stroma, urokinasetype plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPAproducing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumorderived human breast fibroblasts to produce uPA protein and the myofibroblast marker smoothmuscleactin (SMA) in response to various cytokines implicated in the process of tissueremodeling during malignant transformation.We found that fibroblasts produced increased amounts of uPA protein after exposure to aFGF, bFGF, EGF, PDGFBB, and IFN, were unaffected in this respect by IL6, MCSF, GMCSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL1, TNF, IGFI, and IGFII. None of these cytokines were able to induce a striking increase in the fraction of SMApositive fibroblasts. On the other hand, 25pM TGF₁ increased the fraction of SMApositive fibroblasts 5fold in both normal and tumortissuederived fibroblasts. Nonetheless, the normalderived fibroblasts were unaffected in their uPAproducing capacity by TGF₁, and the tumorderived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basaluPAproduction of both normal and tumorderived fibroblasts was increased by autocrinely produced bFGFlike activity, and that the basaluPAproduction of at least the normalderived fibroblasts was decreased by autocrinely produced IGFlike activity.Altogether, our data suggest an active role for fibroblasts in the process of uPAdirected breast tumor proteolysis.