In vivo stimulation by tamoxifen of cathepsin D RNA level in breast cancer

We have previously shown that 3 weeks of treatment with tamoxifen, of patients with primary breast carcinomas, increased cytosolic cathepsin D protein in oestrogen receptor (ER) positive tumours [Maudelonde et al., Cancer 1989, 63, 1265-1270]. In order to investigate the mechanism of this increase and to eliminate a transient flare-up effect, we semi-quantified cathepsin D RNA levels by in situ hybridisation in 32 breast carcinomas from patients treated with tamoxifen for 3 weeks prior to surgery and in 35 breast cancer patients receiving no tamoxifen. We found that tamoxifen increased cathepsin D RNA level regardless of the ER status of the tumours. In ER positive tumours, tamoxifen increased the cathepsin D RNA level to the same extent as cytosolic cathepsin D protein but not in ER negative tumours. The induction of cathepsin D RNA by tamoxifen in ER positive tumours was probably due to its agonist activity, also observed in vitro in breast cancer cell lines. These results suggest that the cathepsin D gene is inducible by oestrogens in ER positive breast cancer as it is in breast cancer cell lines.     

Sensitization to doxorubicin resistance in breast cancer cell lines by tamoxifen and megestrol acetate

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.     

p53 mutation and tamoxifen resistance in breast cancer

A substantial portion of patients with estrogen receptor-positive breast cancer fail to respond to estrogen depletion or to the antiestrogen tamoxifen. The molecular changes that lead to tamoxifen resistance and estrogen-independent growth are unknown. To test the hypothesis that a p53 mutation could result in tamoxifen resistance and estrogen-independent growth, the MCF-7 cell line was transfected with p53 cDNA which was mutated at codon 179 (histidine to glutamine). MCF-7 is an estrogen receptor-positive, estrogen-dependent, tamoxifen-sensitive cell line with only wild-type p53. The presence of transfected mutant p53 cDNA was verified by the PCR, and overexpression of p53 protein was assessed by Western blotting. Five separate mutant-transfected clones were selected and tested in subsequent growth experiments. In monolayer culture, there was no consistent evidence of estrogen-independent growth or tamoxifen resistance in the mutant transfectants compared with vector-only controls or the parental cell line. In soft agar growth experiments, four of five mutant transfectants remained sensitive to tamoxifen in a dose-dependent manner. In the presence of wild-type p53, mutant 179 p53 protein does not result in estrogen-independent growth or tamoxifen resistance. These results do not exclude the possibility that other p53 mutational types could result in tamoxifen resistance, or that loss of the remaining wild-type allele may be necessary to result in this phenotype.     

Effects of tamoxifen in relation to breast cancer

It has been suggested that tamoxifen possesses estrogenic properties which may induce regression of mammary tumors in humans. However, it has been shown that tamoxifen inhibits the estradiol-stimulated changes in immature, ovariectomized, and mature rats. Whereas estradiol stimulates cell division, tamoxifen produces endometrial hypertrophy, even though it has been shown to bind to cytoplasmic estrogen receptors. In vitro experiments with human breast cancer cells have shown that tamoxifen has effects other than that of a simple estrogen antagonist. It is concluded that in addition to its antiestrogneic properties, it may act as a partial or a typical estrogen agonist, which may account for its antitumor activity in postmenopausal patients with breast cancer.     

Clomiphene analogs with activity in vitro and in vivo against human breast cancer cells

The masking procedure by Paradiso and Nakayama (1991) (Vision Research, 31, 1221-1236) was used to investigate brightness filling-in within textures made of line elements: a texture stimulus was masked by a second stimulus containing a square contour. When a uniform texture was presented, the texture region inside the masking square appeared darkened and a small number of texture elements were perceived with a degenerated shape, appearing as dim dots or shorter line elements; it is as if the line element expanded from a bright point to fill the entire region defined by its contour. If the texture stimulus was a texture patch segregating from the surrounding texture by an orientation gradient and this patch was inside the square mask, darkening was not as strong as in the previous condition, and masked line elements preserved their elongated shape. Brightness spreading was measured in two experiments using dichoptic presentations. Experiment 1 used an adjustment task and showed that the brightness of texture line elements spread from equiluminant borders between segregating textures. Experiment 2 used a matching task and demonstrated that spreading was blocked by segregation borders dependent on the orientation gradient between texture line elements. The selectivity for line orientation began 40-80 msec after texture onset and maximal spreading occurred at approximately 120 msec. These findings may indicate that two processes subserve filling-in within textures: the first spreads isotropically the mean stimulus luminance at an initial processing stage of image analysis; at a later stage, the second spreads a texture flow (both brightness and shape of line elements) directed along the orientation of texture line elements. The texture flow mechanism fills in with a texture surface the region bounded by segregation contours.     

T cell anergy is programmed early after exposure to bacterial superantigen in vivo

We have investigated the effects of the protein synthesis inhibitor, cycloheximide (CHX), on the induction of post-thymic T cell tolerance in mice primed with the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB). A single injection of 1 mg CHX prevented protein synthesis in splenic cells for < 6 h in vivo. The concomitant administration of SEB and CHX prevented induction of SEB-specific anergy, but did not interfere with the deletion of SEB-specific V beta 8+ T cells by activation-induced, programmed cell death. When CHX was given > or = 24 h after SEB administration the expression of anergy was not affected. These findings suggest that anergy and deletion represent independent processes. Furthermore, these observations, together with the fact that SEB retains the potential to induce anergy in specific T cells 8 h after priming in vivo, imply that the determination of alternate fates (anergy or death) occurs at early time points after SEB injection.     

Down-regulation of TSC-22 (transforming growth factor beta-stimulated clone 22) markedly enhances the growth of a human salivary gland cancer cell line in vitro and in vivo

The mouse X-linked mutants lined and stripey are associated with lethality of affected males in utero and a striping of the coat in carrier females. We demonstrate that the underlying mutations are nested deletions which lie in the Phex-Amelx chromosomal segment conserved between man and mouse. The lined deletion contains less than approximately 0.7 cM of genetic material and includes the growth factor-regulated protein kinase gene, Rsk2. Stripey carries a larger deletion which removes approximately 2.0 cM of genetic material, including Rsk2 and the pyruvate dehydrogenase E1alpha subunit gene, Pdha1 . Since Coffin-Lowry syndrome and neonatal lactic acidosis are associated with mutations in the human homologues of Rsk2 and Pdha1 respectively, lined and stripey provide models for gene deficiencies in these disorders.     

Prolonged exposure to halothane and associated changes in carbohydrate metabolism in rat muscles in vivo

Halothane, an anesthetic presently used in animal experimentation, is reported to stimulate glycogen breakdown in isolated preparations of rat skeletal muscles, suggesting that it may not be a suitable anesthetic for the study of glycogen metabolism in rats in vivo. The purpose of this study was to establish whether prolonged exposure to halothane in rats in vivo is associated with accelerated glycogenolysis. Exposure of rats to halothane for up to 1 h was not accompanied by either any change in the levels of glycogen or increase in activity ratios of glycogen phosphorylase in muscles, irrespective of their fiber compositions. In marked contrast, the levels of lactate, inorganic phosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-bisphosphate, and fructose 2, 6-bisphosphate changed progressively during anesthesia. Accordingly, the interpretation of muscle metabolite levels must be performed with caution in experiments involving prolonged exposure to halothane. Overall, our findings indicate that the reported halothane-mediated stimulation of glycogen breakdown in vitro is likely to be an artifact and that halothane is a suitable anesthetic for experiments concerned with glycogen metabolism in rats.     

ADP-ribosylation of brain neuronal proteins is altered by in vitro and in vivo exposure to inorganic mercury

ADP-ribosylation is an essential process in the metabolism of brain neuronal proteins, including the regulation of assembly and disassembly of biological polymers. Here, we examine the effect of HgCl2 exposure on the ADP-ribosylation of tubulin and actin, both cytoskeletal proteins also found in neurons, and B-50/43-kDa growth-associated protein (B-50/GAP-43), a neuronal tissue-specific phosphoprotein. In rats we demonstrate, with both in vitro and in vivo experiments, that HgCl2 markedly inhibits the ADP-ribosylation of tubulin and actin. This is direct quantitative evidence that HgCl2, a toxic xenobiotic, alters specific neurochemical reactions involved in maintaining brain neuron structure.     

Induction of apoptosis by tamoxifen and ICI 182780 in primary breast cancer

Hormonal breast cancer therapies have traditionally been considered cytostatic, but recent pre-clinical data suggest that anti-oestrogens can induce apoptosis. The aim of this study was to assess whether tamoxifen (TAM) and ICI 182780 (ICI) could induce apoptosis in human breast cancer, and whether this was related to oestrogen receptor status. We measured apoptosis in primary breast cancer patients before and after pre-surgical treatment with 20 mg/day TAM (study 1) or 6 or 18 mg/day ICI (study 2). In each study there was a randomised non-treatment (NT) control group. TAM significantly increased apoptotic index (AI) in ER+ but not in ER- tumours. There was a significant increase in AI following treatment with ICI. Insufficient pairs of samples were available to determine whether this change was confined to ER+ tumours, but in a cross-sectional analysis AI was significantly higher in excision biopsies for ICI-treated than NT patients for ER+ but not ER- tumours. Our results provide clinical evidence that apoptosis may be induced in ER+ primary breast cancer by both non-steroidal and steroidal anti-oestrogens.     

Organochlorine exposure and risk of breast cancer

BACKGROUND: Some organochlorine compounds may have weak oestrogenic effects and are, therefore, suspected of increasing the risk of breast cancer. We assessed prospectively the risk of breast cancer in relation to serum concentrations of several organochlorine compounds. METHODS: In 1976, serum samples from 7712 women were obtained from participants in the Copenhagen City Heart Study as part of physical examinations and interviews about lifestyle factors. During 17 years of follow-up, 268 women developed invasive breast cancer. Each woman with breast cancer was matched with two breast-cancer-free women from the remaining cohort. We analysed in 1996-97 the serum samples from 240 women with breast cancer and 477 controls. FINDINGS: Dieldrin was associated with a significantly increased dose-related risk of breast cancer (adjusted odds ratio 2.05 [95% CI 1.17-3.57], p for trend 0.01). Beta-hexachlorocyclohexane increased risk slightly but not significantly (p for trend 0.24). There was no overall association between risk of breast cancer and p,p'-dichlorodiphenyltrichloroethane or metabolites or for polychlorinated biphenyls. Exclusion of women with breast cancer diagnosed within 5 years of blood sampling strengthened the result for dieldrin, but did not affect the other results. INTERPRETATION: These findings support the hypothesis that exposure to xeno-oestrogens may increase the risk of breast cancer.     

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.     

In vitro and in vivo effects of cisplatin and etoposide in combination on small cell lung cancer cell lines

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isobologram analysis. Used together, CDDP and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC-3), additive effects on 6 (SBC-2, SBC-5, Lu130, Lu134AH, Lu135T and H69) and an antagonistic effect on 1 (SBC-1). In the in vivo experiment, nude mice were inoculated with SBC-1, SBC-3 and SBC-5 cells. Two or 5 mg/kg CDDP and 10 or 30 mg/kg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may be attributable not to drug interaction at the cellular level but to the feasibility of combined use of them at full doses without overlapping side effects.     

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.     

Effect of MRC-5 fibroblast conditioned medium on breast cancer cell motility and invasion in vitro

OBJECTIVE: To study peritoneal fluid and solute transport characteristics using different polyglucose solutions with and without the addition of glucose. DESIGN: Thirty-one rats were divided into three groups. A 4-hour dwell study with frequent dialysate and blood samples was performed in each rat using 25 mL of 7.5% polyglucose solution (PG, n = 11), 7.5% polyglucose + 0.35% glucose solution (PG1, n = 12), or 3.75% polyglucose + 1.93% glucose solution (PG2, n = 8). Radiolabeled human albumin (RISA) was added to the solutions as an intraperitoneal volume (i.p.V) marker. In addition, polyglucose degradation was evaluated ex vivo over 24 hours. EXPERIMENTAL ANIMALS: Thirty-one male Sprague-Dawley rats (300 g) were used. MAIN OUTCOME MEASURES: Fluid and solute (glucose, urea, sodium, potassium, and total protein) transport characteristics as well as changes in dialysate osmolality were evaluated. RESULTS: The i.p.V was higher in the PG1 and PG2 groups than in the PG group during the first 2 hours of the dwell. The i.p.V, in fact, decreased during the first hour of the dwell in the PG group. However, the net ultrafiltration at 4 hours tended to be lower in the PG2 (3.2 +/- 1.5 mL) group compared to the PG (5.1 +/- 2.3 mL) and the PG1 groups (5.2 +/- 2.1 mL) (p = 0.07), and no significant difference was found between the PG and PG1 groups. Adding glucose to the PG solution increased the RISA elimination rate (KE, representing the fluid absorption rate from the peritoneal cavity): 25.5 +/- 8.2, 37.5 +/- 12.2, and 42.5 +/- 8.9 microL/min for the PG, PG1, and the PG2 group, respectively, p < 0.01. Dialysate osmolality (D[OS]) increased with the dwell time in the PG and PG1 groups but decreased in the PG2 group.The increase in D(OS) was partially due to the degradation of glucose polymer, which was supported by the marked increase in osmolality over 24 hours of incubation of PG solution with peritoneal fluid, ex vivo. The diffusive mass transport coefficient for the investigated solutes did not differ among the three groups (except for glucose, which was significantly lower in the PG group). The sieving coefficient for sodium was significantly higher in the PG group compared to the PG1 group (p < 0.05). CONCLUSION: Our results suggest that, although there was an initial decrease in the intraperitoneal dialysate volume, significant amounts of fluid can be removed by polyglucose solution during a single 4-hour dwell in rats, despite the low osmolality of the solution. The positive fluid removal induced by the PG solution is partially due to the lower fluid absorption rate associated with this solution and may, to some extent, also be due to the degradation of glucose polymer within the peritoneal cavity, resulting in increased dialysate osmolality. The addition of glucose to the polyglucose solution does not seem to improve ultrafiltration in a 4-hour dwell in the rat model. However, the peritoneal fluid absorption rate may be increased, and peritoneal transport of glucose and sodium may be altered, by adding glucose to the polyglucose solution.     

Molybdenum, xanthine oxidase and riboflavin levels in tamoxifen treated postmenopausal women with breast cancer

40 cases postmenopausal women with breast cancer constituted the study group and 20 sex and age matched formed the control group. The study group of untreated patients showed nonsignificant decrease in molybdenum but significant decrease in blood xanthine oxidase and riboflavin levels. Tamoxifen treated patients showed nonsignificant increase in molybdenum, after 3 months, significant increase after 6 months and significant increase in xanthine oxidase and riboflavin levels. Thus tamoxifen when given in breast cancer helps in amelioration of the diseased condition.     

CD4+ lymphocytes provide MUC1-specific tumor immunity in vivo that is undetectable in vitro and is absent in MUC1 transgenic mice

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC .Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.     

Response-specific antiestrogen resistance in a newly characterized MCF-7 human breast cancer cell line resulting from long-term exposure to trans-hydroxytamoxifen

To understand better the antiestrogen-resistant phenotype that frequently develops in breast cancer patients receiving tamoxifen, we cultured MCF-7 breast cancer cells long-term (>1 yr) in the presence of the antiestrogen trans-hydroxytamoxifen (TOT) to generate a subline refractory to the growth-suppressive effects of TOT. This subline (designated MCF/TOT) showed growth stimulation, rather than inhibition, with TOT and diminished growth stimulation with estradiol (E2), yet remained as sensitive as the parental cells to growth suppression by another antiestrogen, ICI 164,384. Estrogen receptor (ER) levels were maintained at 40% of that in parent MCF-7 cells, but MCF/TOT cells failed to show an increase in progesterone receptor content in response to E2 or TOT treatment. In contrast, the MCF/TOT subline behaved like parental cells in terms of E2 and TOT regulation of ER and pS2 expression and transactivation of a transiently transfected estrogen-responsive gene construct. DNA sequencing of the hormone binding domain of the ER from both MCF-7 and MCF/TOT cells confirmed the presence of wild-type ER and exon 5 and exon 7 deletion splice variants, but showed no point mutations. Compared to the parental cells, the MCF/TOT subline showed reduced sensitivity to the growth-suppressive effects of retinoic acid and complete resistance to exogenous TGF-beta1. The altered growth responsiveness of MCF/TOT cells to TOT and TGF-beta1 was partly to fully reversible following TOT withdrawal for 16 weeks. Our findings underscore the fact that antiestrogen resistance is response-specific; that loss of growth suppression by TOT appears to be due to the acquisition of weak growth stimulation; and that resistance to TOT does not mean global resistance to other more pure antiestrogens such as ICI 164,384, implying that these antiestrogens must act by somewhat different mechanisms. The association of reduced retinoic acid responsiveness and insensitivity to exogenous TGF-beta with antiestrogen growth resistance in these cells supports the increasing evidence for interrelationships among cell regulatory pathways utilized by these three growth-suppressive agents in breast cancer cells. In addition, our findings indicate that one mechanism of antiestrogen resistance, as seen in MCF/TOT cells, may involve alterations in growth factor and other hormonal pathways that affect the ER response pathway.