Multiple actions of synthetic ‘progestins’ on the growth of ZR-75-1 human breast cancer cells: Anin vitro model for the simultaneous assay of androgen,progestin, estrogen,and glucocorticoid agonistic and antagonistic activities of steroids

This study was designed to assess the multiple steroid receptor mediated activities of a series of synthetic progestins on breast cancer cell growth, using the human ZR-75-1 cell line which possesses functional estrogen (ER), androgen (AR), and glucocorticoid (GR) receptors as well as progesterone (PgR) receptors. Four 17-hydroxyprogesterone derivatives (chlormadinone acetate, CMA; cyproterone acetate, CPA; medroxyprogesterone acetate, MPA; and megestrol acetate, MGA) and two 19-nortestosterone derivatives (norethindrone, NRE, and norgestrel, NRG) were thus investigated.Based on the requirement of estrogens for PgR-mediated antiproliferative effects and the reversal of PgR-mediated action by insulin, it was found that although all progestins could inhibit ZR-75-1 cell growth through the PgR at low concentrations, the relative contribution of this receptor in cell growth control is highly variable between compounds. The quantitative importance of PgR-mediated inhibition of cell proliferation was inversely related to the amplitude of the androgenic effects induced by the compounds, the AR-mediated effects increasing in the order CPA < MGA < CMA < NRE < NRG < MPA. The specificity of these androgenic effects is further supported by their reversal upon addition of the antiandrogen hydroxyflutamide. In addition, the 17-hydroxyprogesterone derivatives, but not the 19-nortestosterone derivatives, had glucocorticoid activities at high (micromolar) concentrations, as shown by reversal of growth inhibition by the antagonist RU486 in the presence of saturating concentrations of 5-dihydrotestosterone. All progestins tested, except MPA and NRE, also had some antiglucocorticoid activity, NRG being the most potent in this respect. Finally, NRE and NRG exerted a marked mitogenic effect in estrogen-free medium which was clearly mediated through the ER as shown by the competitive reversal of their action by the steroidal antiestrogen EM-139.The present results show that growth measurements of the human breast cancer cells ZR-75-1 permit, with the appropriate steroid additions, the assay of progestin, androgen, estrogen, and glucocorticoid agonistic as vell as antagonistic activities of test compounds. The present study shows, somewhat surprisingly, that while the AR is almost completely responsible for the action of MPA at low concentrations, the majority of the action of NRE, NRG, and MGA is also exerted through AR, while the androgenic action of CPA plays a lower role in the growth inhibition induced by this compound. Such a model should be of great help in designing more specific steroid drugs and in better understanding the role of the different steroid classes which can be used to control the growth of hormone-sensitive cancer. The present data also indicate that progestin is an inappropriate name for MPA, NRE, NRG, MGA, CMA, and CPA, which all possess other and sometimes more potent steroidal activites than those related to interaction with the progesterone receptor.Abbreviations CMA chlormadinone acetate [17-acetoxy-6-chloropregna-4, 6-dien-3, 20-dione] - CPA cyproterone acetate [17-acetoxy-6-chloro-1,2-methylene-pregna-4, 6-dien-3, 20-dione] - DEX dexamethasone [9-fluoro-11, 17, 21-trihydroxy-16-methyl-pregna-1, 4-dien-3, 20-dione] - DHT 5-dihydrotestosterone [17-hydroxy-5-androstan-3-one] - E₂ estradiol [estra-1, 3, 5 (10)-trien-3, 17-diol] - EM 139 [N-n-butyl-N-methyl-11-(16-chloro-3, 17-dihydroxyestra-1, 3, 5 (10)-triene-7-yl) undecanamide] - MGA megestrol acetate [17-acetoxy-6-methylpregna-4, 6-dien-3, 20-dionel] - MPA medroxyprogesterone acetate [17-acetoxy-6-methylpregn-4-en-3, 20-dione] - NRE norethindrone [17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one] - NRG norgestrel [13-ethyl-17-hydroxy-18, 19-dinor-17-pregn-4-en-20-yn-3-one] - OHF hydroxyflutamide (SCH 16423) [, , -trifluoro-2-methyl-4-nitro-m-lactotoluidide] - R1881 methyltrienolone [17-hydroxy-17-methyl estra-4, 9, 11-trien-3-one] - R5020 promegestone [17, 21-dimethyl-19-norpregna-4, 9-dien-3, 20-dione] - RU486 [17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-estra-4, 9-dien-3-one] - triamcinolone acetonide [9-fluoro-11, 21-dihydroxy-16, 17(1-methylethylidenebis oxy) pregna-1, 4-dien-3, 20-dione]     

Effect of Dose and Infusion Time on the Delivery of p-boronophenylalanine for Neutron Capture Therapy

Clinical trials of boron neutron capture therapy (BNCT) for glioblastoma multiforme are currently in progress using p-boronophenylalanine (BPA) as the 10B delivery agent. Enhancement of tumor boron uptake and/or the tumor-to-blood (T:B) boron concentration ratio would have the potential of significantly improving the therapeutic gain of BNCT. The effects of total dose, infusion time, and route of administration of BPA on tumor and blood boron concentrations were studied in rats bearing the 9L gliosarcoma. Increasing the total dose of BPA from 250 to 1000mg/kg, administered intravenously over a 2-h infusion period, resulted in an increase in tumor boron concentration from 30 to 70µg 10B/g, with a constant T:B boron concentration ratio of about 3.7:1. Similarly, extension of the infusion time from 2 to 6h, at a constant dose-rate of 125mg BPA/kg/h, resulted in an increase in tumor boron concentration from 30 to 80µg 10B/g, while, again, maintaining a constant T:B ratio of about 3.7:1. In contrast, intracarotid infusion of BPA for 1h at a dose rate of 125mg BPA/kg resulted in an increase in the tumor boron concentration from 26 to 38µg 10B/g with a corresponding increase in the T:B ratio from 3.5:1 to 5.0:1. The effects of these results on the therapeutic gain potentially achievable with BNCT are discussed.     

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n=10), no treatment; Group 2 (n=9), leuprolide (100g/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n=10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833g/day over a 60-day period. Group 4 (n=10), leuprolide (100g/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n=9), oophorectomy two weeks prior to DMBA with 0.05mg of estradiol in depot form, releasing 0.833g/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n=10), leuprolide (100g/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n=10), leuprolide (100g/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n=9), leuprolide (100g/kg/day) and bromocriptine (83g/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.     

Granulocyte colony-stimulating factor-producing malignant pleural mesothelioma with the expression of other cytokines

We report a 65-year-old man with malignant pleural mesothelioma that produced granulocyte colony-stimulating factor (G-CSF) and other cytokines. At the first presentation, the WBC count was 8600 cells/µl and C-reactive protein (CRP) was 0.6mg/dl. Seventeen months later, the WBC count had increased to 53600 cells/µl (93% neutrophils) and CRP to 27.1mg/dl. The serum concentration of G-CSF had increased to 36.0pg/dl (normal range, <5.0pg/dl), interleukin 1 (IL-1) to 46.0pg/dl (normal range, <10pg/dl), and IL-6 to 197pg/dl (normal range, <4.0pg/dl). The patient died 19 months after the first presentation, and 6 weeks after sudden leukocytosis. At autopsy, a diagnosis of malignant pleural mesothelioma was made. The tumor cells were positive for anti-human G-CSF, granulocyte-macrophage colony-stimulating factor, IL-1, and IL-6 antibodies. Malignant mesothelioma may produce G-CSF and other cytokines. Mesothelial cells may have the potential to produce G-CSF and other cytokines in the progress of malignant formation, and this may be a factor influencing the poor prognosis.     

Ultrastructural Changes of the Vascular Endothelium after Intra-arterial Administration of Tumor Necrosis Factor-alpha (TNFα) in Rat Gliomas

The ensuing ultrastructural changes in tumor vascular endothelial cells following intra-arterial administration of tumor necrosis factor- (TNF) were studied in an experimental rat glioma model. C6 glioma cells were implanted in Wistar rats and then after 14 days, 5×10³U of human natural-type TNF (1.7×10⁵U/m²) was administered through the carotid artery. The animals were sacrificed at 3 or 24h after TNF treatment. A detailed examination with transmission electron microscope revealed swelling of the tumor vascular endothelial cell nuclei and mitochondria with matrix densities at 3h. At 24h, these cells demonstrated the presence of high amplitude mitochondrial swelling or the violent blebbing characteristic of damaged mitochondria; the cytoplasm was swollen enormously and there were dissolution of cytoplasmic organelles and rupture of the plasma membrane. The observed findings were typical of cell necrosis and confirms yet another mechanism by which TNF exerts its anti-tumor effects, that is, necrotizing effects on tumor vascular endothelium. The information appears to be important in the context of clinical application of intra-arterial TNF in the treatment of malignant gliomas.     

Infertility and risk of fatal ovarian cancer in a prospective cohort of US women

Objectives: It is difficult to separate the possible role of fertility drugs from underlying infertility as risk factors for ovarian cancer. The present study examined the relationship between self-reported infertility and death from ovarian cancer among married women unlikely to have been exposured to fertility drugs.Methods: Women were selected for study from the 676,526 female participants in Cancer Prevention Study II (CPS-II). After twelve years of follow-up, 797 deaths from ovarian cancer were observed among women with no prior history of cancer or hysterectomy and 40 years of age or older in 1967 when ovulatory stimulants were approved in the United States. Cox proportional hazards modeling was used to compute rate ratios (RRs) and to adjust for other potential risk factors.Results: Overall, self-reported infertility was not significantly associated with ovarian cancer mortality (adjusted rate ratio (RR)=1.1, 95 percent confidence interval (CI)=0.9-1.3). Ovarian cancer death rates among nulligravid women with self-reported infertility, however, were 40 percent higher than for nulligravid women who never tried to become pregnant (RR=1.4, 95 percent CI=0.9-2.4). Multigravid women who reported infertility problems were not at increased risk.Conclusions: These results suggest that infertility itself, without concomitant exposure to fertility drugs, may increase risk of fatal ovarian cancer among nulligravid women.     

Prognostic relevance of transforming growth factor alpha (TGF-α) and tumor necrosis factor alpha (TNF-α) detected in breast cancer tissues by immunohistochemistry

Summary The function of different growth factors in the development and progression of malignant tumors and the role of cytotoxic cytokines in the host response generated against neoplasms have been recently studied. Anti-TGF- and anti-TNF- monoclonal antibody families have been developed and characterized previously by our laboratory. Libraries of anti-TGF- and anti-TNF- monoclonal antibodies were selected for equal immunoreactivity both in native (frozen) and in formaldehyde fixed, paraffin embedded histological sections. No differences were found between native and fixed samples demonstrated in 10 cases in the present prospective study. Retrospective investigation was performed in 35 histopathological specimens of breast cancer patients detailed clinically and observed during 5 years after the surgical treatment. Correlation between TGF- and/or TNF- expression and clinical staging - TNM score, lymph node metastasis, tumor recurrence and survival time - was analyzed. According to our present study, the TGF- positive patients had worse clinical prognosis than the TNF- positive and double positive cases during long term observation.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

Survival and tumor characteristics of German hereditary breast cancer patients

Reports from different countries have been inconclusive in attempting to relate the BRCA1 mutation status to the survival of breast cancer patients. The purpose of this study was to investigate overall and disease-free survival for German hereditary breast cancer patients. Data on clinical outcome and data on age at diagnosis of breast cancer, histology, tumor size, lymph node status, histological grade, and laterality of 36 breast cancer patients from 12 families with a BRCA1 mutation and from one family with strong evidence for linkage to BRCA1 were compared with those of 49 hereditary breast cancer patients from 23 families that did not harbor a BRCA1 mutation. Overall and disease-free survival was estimated for both groups. BRCA1 mutation carriers had a significantly earlier age of diagnosis than non-carriers (p=0.0001) and more frequently developed contralateral breast cancer (p=0.04). Also, BRCA1-associated tumors more frequently were of larger size (p=0.041) and higher grade of malignancy (p=0.005) than non-BRCA1-associated tumors. Whereas no difference in overall survival was seen, disease-free survival at 10 years differed significantly with 53.3% for BRCA1 mutation carriers and 76% for non- carriers (p=0.02). However, after stratification for age and in multivariate analysis for mutation status, age, and bilaterality, it was shown that the worse prognosis for BRCA1 mutation carriers disappeared. Our results suggest that the worse prognosis of BRCA1 mutation carriers in terms of disease-free survival may in large part be due to the age of onset of breast cancer in this population. Thus, BRCA1 mutation status does not appear to be an independent prognostic factor.     

Molecular Targeting with Recombinant Cytotoxins of Interleukin-13 Receptor α 2-expressing Glioma

A restricted receptor for interleukin 13 (IL-13R2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R2, we established an IL-13R2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R2. G-26-IL-13R2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R2 in vivo. These characteristics of the G-26-IL-13R2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50mm³, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

START: A European state-of-the-art on-line instrument for clinical oncologists

START (State-of-the-Art Oncology in Europe), a freely available resource on the Internet, is a European information base of current clinical approaches to human tumours. Its aim is to help clinical oncologists make appropriate clinical decisions by providing them with updated information reflecting state-of-the-art cancer treatment as perceived by the European oncology community. It is based upon contributions from authors and internal reviewers from all over Europe as selected by START's editorial board under the supervision of an advisory board and a scientific committee. Close collaborations with the main European cancer societies are ongoing. An external feedback process augments the mechanisms for rendering START a truly European instrument. START is concerned with evidence-based cancer medicine, and the main clinical options are thus codified and their bases indicated in accord with a scale worked out from the perspective of clinical decision-making. Therapeutic options may be standard, investigational, or suitable for individual clinical use (within the context of a decision made jointly by the patient and the physician). The goal of instruments such as START is to improve the quality of patient care. In addition, START hopes to make contributions to the methodology by which medical research is transformed into clinical decisions.     

Cytotoxicity of ketoconazole in malignant cell lines

Summary The cytotoxic effects of ketoconazole, an antifungal agent known to have some activity against human prostate cancer, adrenal cancer, and male metastatic breast cancer, were evaluated using colony-growth and clonogenic assays in eight malignant cell lines. The cytotoxicity of ketoconazole showed a dose-and time-dependent pattern, with the following concentrations, inhibiting 90% of the growing colonies (IC₉₀): MCF 7 (human breast cancer) 7.25 g/ml, T 47 D (human breast cancer) 9.0 g/ml, MiaPaCa (human pancreatic carcinoma) 10.0 g/ml, COLO 357 (human pancreatic carcinoma) 9.5 g/ml, HCT 8 (human colonic adenocarcinoma) 27.1 g/ml, DU 145 (human prostatic cancer) 40.0 g/ml, AR 42 J (rat pancreatic carcinoma) 9.0 g/ml, and L1210 (murine leukemia) 8.6 g/ml. Since a concentration of 10 g/ml can be achieved in humans, the use of ketoconazole in human malignancies might be worthy of clinical evaluation.This investigation was supported in part by a grant from the German Volkswagen Foundation, Hannover, Federal Republic of Germany and by a gift from Dr Virgil Gianelli, Stockton, California     

Alcoholic beverage consumption and risk of breast cancer in Spain

The relation between alcoholic beverage consumption and risk of breast cancer was examined. We used data from a population-based, case-control study that included almost all incident cases occurring in five Spanish regions from February 1990 to July 1991. A total of 762 women between 18 and 75 years of age, with a histologically confirmed, first diagnosis of breast cancer, were compared with 988 control women. Alcoholic beverage intake was measured by an interviewer-administered, semiquantitative food-frequency questionnaire. We used nondrinkers as the reference category and divided the remainder into four categories according to alcohol intake. The multiple logistic analyses included not only alcohol intake but also possible confounding factors such as total caloric intake, age, socioeconomic status, and reproductive and medical histories. Even at moderate levels of alcohol intake (less than 8 g/day), a 50 percent increase in risk of breast cancer was found. The trend across categories of intake was statistically significant for wine and distilled drinks, as well as total alcohol intake. Consumption of 20 g or more of alcohol per day was associated with a 70 percent elevation in breast cancer risk compared with that of nondrinkers (adjusted relative risk (RR)=1.7,95 percent confidence interval = 1.3–2.3). Although the magnitude of the RR observed in our study was modest, our findings provide further support for a positive association between alcohol consumption and risk of breast cancer.This work was supported by Grant No. 89/0059 from the Spanish Fondo de Investigacion Sanitaria. Dr Martin-Moreno is indebted to the Italian Fondazione per la Formazione Oncologica for awarding him a Paolo Baffi Fellowship in 1991–1992, and to the Spanish Fundacion Mapfre Medicina for a research fellowship award in 1991. Dr Gorgojo is a postdoctoral fellow of the Programa de Formación de Personal del Instituto de Salud Carlos III.     

Quality of life in the first year after breast cancer surgery: rehabilitation needs and patterns of recovery

Background. Although mortality rates from breast cancer are declining, many breast cancer survivors will experience physical and psychological sequelae that affect their everyday lives. Few prospective studies have examined the rehabilitation needs of newly diagnosed breast cancer patients, and little is known about the predictors of healthrelated quality of life (QOL) in this population.Methods. Between 1987 and 1990, 227 women with early stage breast cancer participated in a prospective longitudinal study in which detailed information was collected through interviews, standardized measures of QOL and psychological distress, and clinical evaluation. Comparisons of physical and treatmentrelated problems were made according to type of surgical treatment. Multivariate regression analysis was performed to examine the predictors of QOL at one year after surgery.Results. Physical and treatmentrelated problems were reported frequently one month after breast cancer surgery, and occurred with equal frequency in women receiving modified radical mastectomy or breast conservation treatment. There were no significant differences in problems reported at one year by type of surgery; however, frequently reported problems include numbness in the chest wall or axilla, tightness, pulling or stretching in the arm or axilla, less energy or fatigue, difficulty in sleeping, and hot flashes. There was no relationship between the type of surgery and mood or QOL. Poorer QOL one year after surgery was significantly associated with greater mood disturbance and body image discomfort one month after surgery, as well as positive lymph node involvement. Although the majority of patients experienced substantial disruptions in the physical and psychosocial dimensions of QOL postoperatively, most women recovered during the year after surgery, with only a minority (<10%) significantly worsening during that time.Conclusions. At one year after surgery, most women report high levels of functioning and QOL, with no relationship between the type of surgery and QOL. Women who reported lower levels of QOL at one year after diagnosis had greater mood disturbance and poorer body image one month after surgery, as well as lower income and positive axillary nodes.     

New approach to metabolism of 5′-deoxy-5-fluorouridine in humans with fluorine-19 NMR

Summary The metabolism of 5-deoxy-5-fluorouridine (5dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported -fluoro--alanine. The two major metabolites in urine are unmetabolized 5dFUrd and -fluoro-alanine.     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.     

Coexpression of c-kit and stem cell factor in breast cancer results in enhanced sensitivity to members of the EGF family of growth factors

Insulinlike growth factor (IGF)I protects many cell types from apoptosis. As a result, it is possible that IGFIresponsive cancer cells may be resistant to apoptosisinducing chemotherapies. Therefore, we examined the effects of IGFI on paclitaxel and doxorubicininduced apoptosis in the IGFIresponsive breast cancer cell line MCF7. Both drugs caused DNA laddering in a dosedependent fashion, and IGFI reduced the formation of ladders. We next examined the effects of IGFI and estradiol on cell survival following drug treatment in monolayer culture. IGFI, but not estradiol, increased survival of MCF7 cells in the presence of either drug. Cell cycle progression and counting of trypanblue stained cells showed that IGFI was inducing proliferation in paclitaxeltreated but not doxorubicintreated cells. However, IGFI decreased the fraction of apoptotic cells in doxorubicin but not paclitaxeltreated cells. Recent work has shown that mitogenactivated protein kinase (MAPK) and phosphotidylinositol3 (PI3) kinase are activated by IGFI in these cells. PI3 kinase activation has been linked to antiapoptotic functions while MAPK activation is associated with proliferation. We found that IGFI rescue of doxorubicininduced apoptosis required PI3 kinase but not MAPK function, suggesting that IGFI inhibited apoptosis. In contrast, IGFI rescue of paclitaxelinduced apoptosis required both PI3 kinase and MAPK, suggesting that IGFImediated protection was due to enhancement of proliferation. Therefore, IGFI attenuated the response of breast cancer cells to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis. Thus, inhibition of IGFI action could be a useful adjuvant to cytotoxic chemotherapy in breast cancer.     

Transforming growth factor beta and the cell surface in tumor progression

Summary Type 1 transforming growth beta (TGF-1) is a multifunctional regulator of cellular differentiation, motility and growth. It is capable of inhibiting or stimulating these processes depending on cell type, cell density, culture conditions and TGF-1 concentration. TGF-1 regulates growth, in part, by inducing the expression and secretion of various types of collagen, which participate in the control of cell adhesion and migration, as well as growth. TGF-1 also regulates cell growth by controlling the response to epidermal growth factor (EGF) and other growth factors, in ways that can either decrease or increase their growth-promoting effects. Alterations in both negative and positive growth responses to TGF-1 play important roles in tumor progression. Loss of sensitivity to growth inhibition by TGF-1 can occur as a result of decreased expression of collagen. Acquisition of sensitivity to growth stimulation, and autocrine transformation by TGF-1, are associated with aberrant EGF receptor regulation. Aberrant growth factor receptor regulation by TGF-1 may be mediated by a protein kinase C (PKC)-dependent pathway which inhibits degradation of growth factor receptor/ligand complexes. The evidence reviewed is consistent with a minimal two-step mechanism for autocrine transformation, which involves production of growth factor and enhanced cellular response as a result of aberrant membrane traffic. Defects in membrane traffic regulation may provide an explanation for common alterations in tumor cell response to both multiple growth inhibitors and growth stimulators, and may also suggest novel approaches to cancer chemotherapy.