phosphorus-radioisotopes and Breast-Neoplasms

A review of the literature has shown that in human breast tumours, large signals from phosphomonoesters (PME) and phosphodiesters (PDE) are evident. In serial measurements in 19 patients with breast cancer, a decrease in PME was significantly associated with a stable or responding disease (p = 0.017), and an increase in PME was associated with disease progression. Extract studies have shown PME to comprise of phosphoethanolamine (PEth) and phosphocholine (PCho), with the PEth to PCho ratio ranging from 1.3 to 12. The PCho content of high grade tumours was found to be higher than low grade tumours. In some animal models, changes in PCho have been shown to correlate with indices of cellular proliferation, and spheroid studies have shown a decrease in PCho content in spheroids with smaller growth fractions. A serial study of 25 patients with advanced primary breast tumours undergoing hormone, chemotherapy or radiotherapy treatments, showed that in this heterogenous group there were significant changes in metabolites that were seen during the first 3 weeks (range 2-4 weeks) of treatment, that correlated with volume change over this period, employed here as a measure of response. Changes in PME (p = 0.003), total phosphate (TP) (p = 0.008) and total nucleoside tri-phosphate (TNTP) (p = 0.02) over 3 (+/-1) weeks were significantly associated with response, as were the levels of PME (p<0.001), PDE (p = 0.01), TP (p = 0.001) and TNTP (p = 0.007) at week 3 (+/-1). PME at week 3 (+/-1) was also significantly associated with the best volume response to treatment (p = 0.03). A reproducibility analysis of results from the observation of normal breast metabolism in four volunteers showed a mean coefficient of variation of 25%, after correcting for changes resulting from the menstrual cycle. Reproducibility studies in four patients with breast cancer showed a mean coefficient of variation of 33%, with the reproducibility being better in patients measured on different days (difference in TP was -6%) compared with those measured on the same day (difference in TP was -29%).

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Breast Neoplasms; Female; Humans; Magnetic Resonance Spectroscopy; Middle Aged; Phospholipids; Phosphorus Radioisotopes; Radiotherapy

In this early Phase II study, the authors investigated the efficacy of intratumoral injection of P-32 chromic phosphate in 17 patients with refractory solid tumors or solitary metastases in terms of response rates and overall survival.. Seventeen patients (median age, 60 years) with either cytostatic drug-resistant tumors or tumors known to be primarily chemotherapy-resistant were entered into the study. After sonographic determination of the tumor volume, P-32 chromic phosphate (74-555 MBq) was injected into the central part of the tumor under sonographic guidance. Follow-up investigations included serial scintigraphy, sonographic examinations, and hematologic studies.. Injection of P-32 chromic phosphate into refractory tumors resulted in remarkable regression. The median survival of all patients was 13 months (range, 8-25 months). The response rate was 71% (12 patients). A complete remission was seen in 7 patients (41%), and the rate of partial remissions was 29% (5 patients). However, 5 patients (30%) did not respond to the treatment. In one patient thrombocytopenia was observed, but no other side effects were apparent. Important pathologic and anatomic changes within the tumor tissue were demonstrated in solitary liver metastases of gastrointestinal malignancies excised in second-look operations. In all cases examined, formation of a cyst within the area of central activity, surrounded by a centrifugal necrotic ring and a marginal fibrotic structure, was found.. Lack of persistent systemic or local side effects, as well as noteworthy efficacy, are properties of this optimal regional treatment modality with P-32 chromic phosphate. This modality deserves consideration for further clinical trials.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Carcinoma, Hepatocellular; Chromium Compounds; Digestive System Neoplasms; Drug Resistance, Neoplasm; Female; Head and Neck Neoplasms; Humans; Injections, Intralesional; Lung Neoplasms; Male; Middle Aged; Neoplasms; Phosphates; Phosphorus Radioisotopes; Survival Rate

Malignant pericardial effusion is usually treated only when signs of cardiac tamponade develop. Several methods of treatment have been reported with an overall response rate of approximately 75%. Since our initial study using intrapericardial 32P-colloid instillation as a treatment modality for pericardial effusion demonstrated a significant higher response rate, this study was conducted to further evaluate the efficacy of intrapericardial 32P-colloid in terms of response rates and duration of remissions. Intrapericardial instillation of 185-370 MBq (5-10 mCi) 32P-colloid in 36 patients with malignant pericardial effusion resulted in a complete remission rate of 94.5% (34 patients) whereas two patients did not respond to treatment due to a foudroyant formation of pericardial fluid. The median duration time was 8 months. No side-effects were observed. These results suggest that intrapericardial instillation of 32P-colloid is a simple, reliable and safe treatment strategy for patients with malignant pericardial effusions. Therefore, since further evidence is provided that 32P-colloid is significantly more effective than external radiation or non-radioactive sclerosing agents, this treatment modality should be considered for the management of malignant pericardial effusion.

Topics: Breast Neoplasms; Cardiac Tamponade; Chromium Compounds; Female; Gastrointestinal Neoplasms; Humans; Instillation, Drug; Lung Neoplasms; Lymphoma; Neoplasms; Pericardial Effusion; Phosphorus Radioisotopes; Radiopharmaceuticals

Sixty-seven patients with disseminated cancer were randomly allocated to treatment with continuous closed chest drainage removing all fluid for 72 hours (PD) or pleural drainage for 72 hours with the instillation into the pleural space of radioactive colloidal chromic phosphate (PD + 32P). Forty-nine patients had breast carcinoma, and the remaining 18 patients had other cancers. Four of 49 patients with breast cancer and 13 of 18 with other cancer were dead in 8 weeks from the onset of effusion. In the group of patients with breast cancer PD + 32P controlled the effusion in 12 of 22 (54%) and PD alone in 15 of 30 episodes (50%). In the nonbreast group of patients PD + 32P controlled the effusion in five of six evaluable episodes (83%), and PD alone was successful in two of nine (22%). In 33% of breast cancer patients and 25% of the nonbreast-cancer patients, systemic chemotherapy produced objective remissions. Pleural effusion did not recur in any of these patients.

Topics: Adult; Aged; Breast Neoplasms; Drainage; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasms; Phosphorus Radioisotopes; Pleural Effusion; Prospective Studies

Topics: Aged; Breast Neoplasms; Clinical Trials as Topic; Cyclophosphamide; Female; Fluorouracil; Humans; Lung Neoplasms; Male; Methotrexate; Middle Aged; Neoplasm Metastasis; Neoplasms; Phosphorus Radioisotopes; Remission, Spontaneous; Vincristine

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ERalpha) and retinoic acid receptor alpha (RARalpha), but not ERbeta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the G(alphai2), G(alphai3), G(alphaq), and G(alphall) proteins, and via activation of G(alphai2) proteins, melatonin suppresses forskolin-induced 3',5'-cyclic adenosine monophosphate production, while melatonin activation of G(alphaq), is able to inhibit phospholipid hydrolysis and ATP's induction of inositol triphosphate production in MCF-7 breast cancer cells. Employing dominant-negative and dominant-positive) forms of these G proteins, we demonstrate that G(alphai2) proteins mediate the suppression of estrogen-induced ERalpha transcriptional activity by melatonin, while the G(q) protein mediates the enhancement of retinoid-induced RARalpha transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both G(alphai2) and G(alphaq) proteins.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Cyclic GMP; Estrogens; Female; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Immunoprecipitation; Luciferases; Melatonin; Phosphorus Radioisotopes; Radioimmunoassay; Receptor, Melatonin, MT1; Receptors, Estrogen; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Retinoic Acid Receptor alpha; Transcription, Genetic; Transfection

We report our experience in the use of radionuclides in the treatment of bone metastases in patients with various primary cancers: breast cancer, prostate cancer, lung cancer, etc.. Eighty-seven patients (53 women, 34 men) with bone metastases were treated for pain relief with either 32-P (71 patients) or 89-Sr (16 patients). Fifty-three of the patients had breast cancer, 27--rostate cancer, 6--lung cancer and 1--kidney cancer. The patients were examined for side effects when 32-P was administered perorally and 89-Sr injected intravenously. We also studied the changes in the levels of hemoglobin, white blood cells (WBCs) count and platelets count.. We found a significant decrease in the WBC and platelet count in the patients treated with 32-P (U = 2.20, P < 0.05 and U = 4.57, P < 0.001) one month after the therapy. These parameters showed no significant decrease in the group treated with 89-Sr. The pain, which was the rationale to use the radioactive isotopes, was relieved and the patients restored their previous mobility.. The fact that 32-P alleviated the grave symptom of pain at the relatively weak radiation dose used (2 mCi) is a strong indication that this radiopharmaceutical can be used successfully for such a purpose, although some authors argue against its use in view of the myelosuppresion it causes. This myelosuppression, however, is mild and transient even without treatment and patients could benefit from this adjuvant treatment to manage the pain syndrome. 89-Sr administered intravenously in a dose of 4mCi also relieves pain efficiently but its use is limited by the cost of the quantity needed for 1 patient and for a single dose. The National Health Insurance Fund currently reimburses for a very limited quantity of this substance which makes the cost of the procedure 15 times as expensive as that using radioactive phosphorus.. Using the radiopharmaceuticals 32-P and 89-Sr provides an additional, easy and efficacious means for palliation of cancer patients with bone metastases, especially those who are refractory to percutaneous irradiation.

Topics: Bone Neoplasms; Breast Neoplasms; Female; Humans; Leukocyte Count; Lung Neoplasms; Male; Pain; Pain Measurement; Phosphorus Radioisotopes; Platelet Count; Prostatic Neoplasms; Strontium Radioisotopes; Treatment Outcome

In vivo studies have demonstrated that pentavalent technetium-99m dimercaptosuccinic acid [(99m)Tc-(V)-DMSA] may be a useful tumour imaging agent. Several studies have suggested that (99m)Tc-(V)-DMSA uptake may be related to the structural similarity between the (99m)Tc-(V)-DMSA core and the PO(4)(3-) anion. As phosphate ions enter cells via NaPi cotransporters, we investigated whether (99m)Tc-(V)-DMSA uptake is mediated by NaPi cotransporters. (99m)Tc-(V)-DMSA and phosphate uptake kinetics were compared in three cancer cell lines (MCF-7, G152 and MG-63) under several conditions (with and without sodium and NaPi cotransporter inhibitor and at different pH). Determination of molecular NaPi cotransporter mRNA expression was performed by reverse-transcriptase polymerase chain reaction (Rt-PCR) assay. Results obtained in the presence of NaPi inhibitor, in sodium-free medium and at alkaline pH showed that (99m)Tc-(V)-DMSA accumulation is linked to NaPi cotransporter functionality. MCF-7 and G152 exhibited the same tracer uptake, whereas MG-63 showed the highest phosphate accumulation and the lowest (99m)Tc-(V)-DMSA uptake. These results were in accordance with mRNA NaPi expression, i.e. all cell lines expressed NaPi type III but MG-63 also co-expressed NaPi type I. The total level of NaPi cotransporter was highly correlated with phosphate accumulation, while the level of type III was related to (99m)Tc-(V)-DMSA uptake. We have demonstrated that (99m)Tc-(V)-DMSA uptake is specifically mediated by NaPi type III in cancer cells.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Glioblastoma; Humans; Hydrogen-Ion Concentration; Kinetics; Metabolic Clearance Rate; Neoplasms; Osteosarcoma; Phosphates; Phosphorus Radioisotopes; Potassium Compounds; Radionuclide Imaging; Radiopharmaceuticals; Sodium; Sodium-Phosphate Cotransporter Proteins; Sodium-Phosphate Cotransporter Proteins, Type I; Symporters; Technetium Tc 99m Dimercaptosuccinic Acid

High-density DNA microarrays require automatic feature extraction methodologies and softwares. These can be a potential source of non-reproducibility of gene expression measurements. Variation in feature location or in signal integration methodology may be a significant contribution to the observed variance in gene expression levels.. We explore sources of variability in feature extraction from DNA microarrays on Nylon membrane with radioactive detection. We introduce a mathematical model of the signal emission and derive methods for correcting biases such as overshining, saturation or variation in probe amount. We also provide a quality metric which can be used qualitatively to flag weak or untrusted signals or quantitatively to modulate the weight of each experiment or gene in higher level analyses (clustering or discriminant analysis).. Our novel feature extraction methodology, based on a mathematical model of the radioactive emission, reduces variability due to saturation, neighbourhood effects and variable probe amount. Furthermore, we provide a fully automatic feature extraction software, BZScan, which implements the algorithms described in this paper.

Topics: Algorithms; Animals; Arabidopsis Proteins; Bias; Breast Neoplasms; Carbon-Oxygen Ligases; Cluster Analysis; Densitometry; Discriminant Analysis; DNA, Neoplasm; DNA, Plant; Gene Expression Profiling; Humans; Image Processing, Computer-Assisted; Mice; Nylons; Oligonucleotide Array Sequence Analysis; Phosphorus Radioisotopes; Polymerase Chain Reaction; Reproducibility of Results; RNA, Messenger; Signal Processing, Computer-Assisted; Software

Nylon membrane-based macroarrays form a widely available alternative to microarrays for the collection of large-scale gene expression data. To carry out repetitive hybridization experiments with nylon cDNA arrays, we used phosphorothioate 33P-cDNA, followed by stripping under relatively mild conditions. We were able to use the same membranes more than 10 times without a measurable reduction in their performance. Thus, our protocol allowsfor more comparative studies of multiple data sets obtained from sequential hybridizations of the same set of membranes. We demonstrate how to analyze repetitive macroarray experiments and to determine the reliability or statistical significance of the gene expression data obtained. Both the averaging of signals per gene and the reversal of nylon membranes had a favorable effect on accuracy. By self-self comparisons, we show that in a duplicate experiment with four membranes, a 2-fold change in the gene expression can be measured reliably.

Topics: Breast Neoplasms; DNA Probes; Endothelium, Vascular; Equipment Design; False Positive Reactions; Gene Expression; Humans; Membranes, Artificial; Nylons; Oligonucleotide Array Sequence Analysis; Phosphorus Radioisotopes; Quality Control; Reproducibility of Results; Sensitivity and Specificity; Tumor Cells, Cultured; Umbilical Veins

Biochemical markers improve the classification and staging of breast cancer and may refine management decisions if it can be shown that they correlate with accepted prognostic factors or patient outcome. Using phosphorus-31 magnetic resonance spectroscopy ((31)P MRS), we determined the phospholipid content of 43 malignant breast tumors, correlating the profiles with specific histopathologic and clinical features and hormone receptor status. Among the 14 phospholipids identified, the mean mole percentage of sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and alkylacylphosphatidylcholine predicted cellular infiltration, infiltration type, elastosis, lymphatic invasion, perineural invasion, necrosis, and estrogen receptor positivity. (31)P MRS phospholipid profile data provide statistical correlations among histologic features and molecules known to play important roles in cellular communication, regulation, and processes unique to malignant tissues.

Topics: Breast Neoplasms; Granulocytes; Humans; Lymphatic Metastasis; Magnetic Resonance Imaging; Multivariate Analysis; Necrosis; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Phosphorus Radioisotopes; Receptors, Estrogen; Sphingomyelins

The effects of cPrG x HCl and epirubicin on the suppression of cell growth were examined on human breast cancer cell line (MDA-MB-231). Either cPrG x HCl or epirubicin alone showed a tumor growth inhibition in a time- and dose-dependent manner, however, the combinatory use of cPrG x HCl together with epirubicin resulted in prominent synergistic effects on the breast cancer cells. In the in vitro studies, the combinatory use of these two drugs accelerated apoptotic cell death as revealed by morphological changes as well as by the appearance of subG1 population by flow cytometry. In addition, confocal microscopy revealed that the accumulation of epirubicin in nucleus increased apparently when cPrG x HCl were present. In the in vivo assay, nude mice bearing xenografted tumor cells received 4 weeks of intraperitoneal administration of cPrG-HCl and epirubicin. After 12 days, the combinatory treatment significantly suppressed the tumor growth compared to the controls. The TUNEL staining revealed that tumor cells in cPrG x HCl plus epirubicin-treated mice exhibited a higher apoptotic rate. In addition, 31P-NMR studies on the xenografted tumor revealed that cPrG x HCl lowered tumor pHi (below pH 6.9). while it did not affected muscle pHi. No pathological changes were observed in any intrinsic organs and the serum alanine aminotransferase levels remained within normal limits among the groups. These results suggest that the combinatory use of cPrG x HCl and epirubicin may be useful for breast cancer therapy.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Epirubicin; Female; Flow Cytometry; Humans; In Situ Nick-End Labeling; Indoles; Injections, Intraperitoneal; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Microscopy, Confocal; Phosphorus Radioisotopes; Pyrroles; Transplantation, Heterologous; Tumor Cells, Cultured

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Topics: Breast Neoplasms; Chromosome Mapping; Chromosomes, Human, Pair 16; Fluorescence; Humans; Loss of Heterozygosity; Phosphorus Radioisotopes; Polymerase Chain Reaction

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for >20 years. However, little is known either qualitatively or quantitatively about the relationship between formation and repair of specific MMC-DNA adducts and specific biological outcomes. The goal of this study was to examine formation and removal of specific MMC-DNA adducts in breast cancer cells using a (32)P-postlabeling assay in relation to cytotoxicity and other biological end points. MMC-DNA adducts were measured in cultured human metastatic MDA-MB-435 cells, in the same cells xenografted as a mammary tumor in nude mice, and in metastatic tumor biopsies obtained from human breast cancer patients undergoing MMC-based therapy. MMC adducts corresponding to the CpG interstrand cross-link, the MMC-G bifunctional monoadduct, and two isomers of the MMC-G monofunctional monoadduct were detected in most samples. Despite similarities in the overall patterns of adduct formation, there were substantial differences between the cultured cells and the in vivo tumors in their adduct distribution profile, kinetics of adduct formation and removal, and relationship of specific adduct levels to cytotoxicity, suggesting that the in vivo microenvironment (e.g., degree of oxygenation, pH, activity of oxidoreductases, and other factors) of breast cancer cells may significantly modulate these parameters.

Topics: Aged; Animals; Biopsy; Breast Neoplasms; Clinical Trials as Topic; DNA Adducts; DNA, Neoplasm; Female; Genome; Humans; Mice; Mice, Nude; Middle Aged; Mitomycin; Neoplasm Transplantation; Phosphorus Radioisotopes; Transplantation, Heterologous; Tumor Cells, Cultured

Abundant complex DNA adducts can be detected in human tissues by a combined 32P-postlabelling and high-performance liquid chromatography (HPLC) method. The HPLC profiles reveal a panorama of nuclease P1-resistant human adducts, which are not among the known human DNA adducts and are suspected of being endogenous. Lipid peroxidation-induced DNA adducts and I-compounds are two possible candidates for these adducts. Therefore, we performed two experiments: one was to identify chromatographically the lipid peroxidation-induced adducts among other human adducts with two acrolein- and crotonaldehyde-derived propano adduct standards (Acr-dG3 and Cro-dG1&2) and a structurally unknown adduct (Cro-DNA) derived from crotonaldehyde-treated DNA; and the other was to analyse the adducts in breast tissue from patients with breast cancer and from controls and to compare their behaviour with that of I-compounds in cancerous tissues. In the first experiment, Acr-dG3 and Cro-dG1 were detected in three human lung tissues, at levels ranging from 3.4 to 8.9 (x 10(-8)) and from not detectable to 2.9 (x 10(-8)), respectively. Acr-dG3 and Cro-DNA were detected in three human colon tissues, at levels of 0.2-0.4 (x 10(-8)) and 1.2-3.4 (x 10(-8)), respectively. In the second experiment, adjacent and tumorous breast tissues from 15 patients with breast cancer (of an average age of 33.4 years) and normal breast tissue from 18 controls (of an average age of 57.3) were analysed for the abundant complex adducts. The total adduct levels in the adjacent and tumorous tissues were lower than in the normal tissues (with medians of 8.0, 11.8 and 13.3 (x 10(-7)), respectively). Significant differences in the adduct levels between adjacent or tumorous tissues and normal tissues were observed in three HPLC peaks, and age was significantly associated with three peaks. These results are consistent with our speculation that the abundant adducts are comprised of lipid peroxidation-induced adducts and human homologues of I-compounds.

Topics: Acrolein; Adult; Age Factors; Aged; Aldehydes; Animals; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Colon; DNA Adducts; Female; Humans; Lung; Male; Middle Aged; Phosphorus Radioisotopes; Rats; Tissue Distribution

The etiology of the majority of human breast cancers is unknown; however, oxidative stress and lipid peroxidation have been suggested to play a role in breast carcinogenesis. To address this possibility, DNA adducts induced by malondialdehyde (MDA), an end product of lipid peroxidation, were analyzed in surgical specimens of normal breast tissues of 51 breast cancer patients using the nuclease P1-enhanced version of the 32P-postlabeling assay. Normal breast tissue samples from 28 noncancer patients receiving reduction mammoplasty served as controls. Two previously characterized putative MDA-deoxyadenosine (dA) and one MDA-deoxyguanosine adduct were detected in all tissue samples examined. Normal breast tissues from cancer patients exhibited significantly higher levels of the putative MDA adducts [median (42.5) and range (2.2-202.8) of relative adduct labeling x 10(9) values] than those found in noncancer controls (median, 15.67; range, 2.4-382.1; P = 0.0001, Mann-Whitney U test). Ten of the 51 cancer patients and 1 of the 28 controls were found to contain the putative MDA adducts at the level of > 1/10(7) nucleotides, a frequency comparable to that found in human liver. Age and body mass did not significantly influence the levels of these adducts. However, the presence of a previously detected benzo(a)pyrene-like DNA adduct in the breast tissues was associated with higher levels of the putative MDA-dA adducts in cancer patients (P = 0.012). The level of the putative MDA-dA adducts was significantly lower in smokers and former smokers compared to nonsmokers among cases after adjusting for age, body mass index, and status of the benzo(a)pyrene-like adduct (P = 0.009). Tumor tissues (n = 11) displayed significantly lower levels of the putative MDA adducts (median, 10.2; range, 5.3-20.6) than their corresponding normal adjacent tissues (median, 25.5; range, 10.5-138; P < 0.01). These findings provide evidence that lipid peroxidation products can accumulate in human breast tissues and reach relatively high levels in the breast tissues of women with breast cancer. There seems to be an interaction between these endogenous DNA modifications and carcinogen exposure-induced DNA adducts. Detection and quantitation of the putative MDA-DNA adducts may potentially be a useful tool in the understanding of breast cancer etiology.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Benzo(a)pyrene; Biomarkers, Tumor; Body Mass Index; Breast; Breast Neoplasms; Carcinogens; Deoxyadenosines; Deoxyguanosine; DNA Adducts; Female; Humans; Lipid Peroxidation; Liver; Malondialdehyde; Middle Aged; Nucleotides; Oxidative Stress; Phosphorus Radioisotopes; Smoking; Smoking Cessation

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptor's affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Casein Kinase II; Cell Line; Chymotrypsin; DNA; DNA-Binding Proteins; Female; Humans; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Estrogen; Recombinant Proteins; Serine; Spodoptera; Substrate Specificity; Transfection; Trypsin; Tumor Cells, Cultured

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

Topics: Adenosine Triphosphate; Animals; Antisense Elements (Genetics); Blotting, Northern; Breast Neoplasms; Female; Genes, erbB-2; Humans; Immunoglobulin A; Immunoglobulin Heavy Chains; In Vitro Techniques; Mice; Phosphorus Radioisotopes; Plasmacytoma; RNA, Messenger; RNA, Neoplasm; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

It has been shown that 17 beta-estradiol stimulates the cycle of phospholipid conversion in the breast tumor cells. The action of tamoxifen on antiestrogen cells is not limited by weakening the stimulating effect of 17 beta-estradiol on the exchange of intracellular phospholipids, but gives rise to a more complicated pattern of changes: inhibited incorporation of 32-P-phosphatidylcholine (PC) and activated exchange of phosphoinositides (PI). The experimental findings of 53 breast tumors have indicated that in 47.2% of cases Tamoxifen alters the PC/PI ratio and causes its 2-fold increase. Such alterations have been found to be induced by the ability of Tamoxifen to suppress the activity of protein kinase C that regulates the synthesis of PC and PI. It is suggested that the revealed capacity of Tamoxifen to change the rate of intracellular phospholipid conversion might be used for evaluating the efficiency of this agent on malignant tumors.

Topics: Autoradiography; Breast Neoplasms; Estradiol; Female; Humans; Phosphatidylcholines; Phosphatidylinositols; Phospholipids; Phosphorus Radioisotopes; Protein Kinase C; Tamoxifen; Tumor Cells, Cultured

Topics: Breast Neoplasms; Cell Line; Cell Nucleus; Cell-Free System; Humans; Indicators and Reagents; Phosphorus Radioisotopes; Plasmids; Progesterone; Radioisotope Dilution Technique; RNA, Neoplasm; TATA Box; Templates, Genetic; Transcription, Genetic; Tumor Cells, Cultured; Uridine Triphosphate

A quantitative method of polymerase chain reaction (PCR) using both digoxigenin and radioactive labelled probes has been used for the detection of the c-erbB-2 proto-oncogene amplification in breast carcinomas with formalin-fixed paraffin-embedded tissue sections. c-erbB-2 proto-oncogene amplification has been demonstrated in 14 infiltrating ductal carcinomas. The technique consisted of the co-amplification of c-erbB-2 and IFN-gamma (interferon-gamma) genes. The latter was considered as a single copy gene per genome-equivalent. The aim of this study was to compare two quantitative PCR techniques based on the incorporation of either digoxigenin-11-dUTP or 32P-dCTP, during amplification. For the colorigenic method, using the Dig system, after electrophoresis and transfer, the specific bands were revealed with a chromogenic substrate of phosphatase. Their intensity estimated by scanning photometry following blot transparisation. After electrophoresis, the radioactive gel was submitted to radioautography and the band intensities evaluated by scanning spectrophotometry. For the 14 samples, a good agreement between both methods was noted. The colorigenic method is a valuable alternative to radiolabelling due to: i) time saving, ii) reagent conservation, iii) safe manipulation and iv) sensitivity of the same order for both methods.

Topics: Base Sequence; Breast Neoplasms; Carcinoma, Ductal, Breast; Colorimetry; Digoxigenin; DNA Primers; DNA, Neoplasm; Evaluation Studies as Topic; Female; Gene Amplification; Genes, erbB-2; Humans; Interferon-gamma; Molecular Probes; Molecular Sequence Data; Phosphorus Radioisotopes; Polymerase Chain Reaction; Proto-Oncogene Mas

Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) has been identified in the non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.

Topics: Adenosine Triphosphate; Base Sequence; Breast Neoplasms; Cathepsin D; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; DNA-Binding Proteins; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Molecular Sequence Data; Nuclear Proteins; Oligonucleotides, Antisense; Phosphorus Radioisotopes; Polyunsaturated Alkamides; Receptors, Estrogen; Recombinant Fusion Proteins; Sp1 Transcription Factor; Sulfuric Acid Esters; Thymidine Kinase; Transfection; Tumor Cells, Cultured

The cycle of phospholipid turnover has been found to be under the negative control of hormonal cytostatics (progesterone) and under the positive control of proliferation stimulants (17 beta-estradiol, epidermal growth factor). Specific changes in the synthesis of phospholipids are shown when tamoxiphen, an antiestrogen and an inhibitor of protein kinase C, was used. The findings suggest that changes in the turnover rate of phospholipids are one of the key stages of steroid action on target cells and may be regarded as an additional criterion of tumor genetic sensibility.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Protein Kinase C; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

Protein tyrosine kinase (PTK) activity was assayed in cytosolic extracts from normal breast tissue, benign tumors, and 84 T1-T2, N0-N1 M0, breast carcinomas. Normal breast tissue extracts yielded an average value of 1.9 +/- 1.1 pmol 32P incorporated/min/mg protein, whereas a mean of 12.5 +/- 6.1 was obtained for cancer samples. With a median follow-up of 34 months, in the series of 40 patients classified N-, PTK positive patients presented a significantly smaller 3-year disease free survival than the PTK negative ones. Multivariate analysis shows that PTK activity emerges as a potential prognostic factor in breast cancer (p = 0.02). These preliminary results will be updated on a bigger cohort of patients.

Topics: Adult; Aged; Breast; Breast Neoplasms; Evaluation Studies as Topic; Female; Humans; Middle Aged; Phosphorus Radioisotopes; Pilot Projects; Predictive Value of Tests; Prognosis; Protein-Tyrosine Kinases; Receptors, Estrogen; Receptors, Progesterone; Statistics as Topic

The in vitro effects of the epidermal growth factor (EGF) and progesterone on phospholipid turnover in cells of 19 human adenocarcinomas (postsurgical material) have been studied. In 58% of tumours EGF increased the 32P incorporation into two basic cell phospholipids--phosphatidylcholine and phosphoinositides. In EGF-insensitive cells progesterone induced no noticeable changes in the basal level of phospholipid metabolism. However, in 10 out of 11 positively responding to EGF adenocarcinomas progesterone inhibited the EGF-dependent activation of 32P incorporation into the phospholipids already on the 15th min after its addition to the cells. Analysis of effects of EGF and the anti-estrogen drug tamoxifen on phospholipid turnover in 22 human mammary tumours did not reveal any significant differences in tamoxifen effect on tumour cells differing in their sensitivity to EGF. Independently of cell sensitivity to EGF, tamoxifen caused some decrease in the 32P incorporation into phosphatidylcholine but increased the label incorporation into phosphoinositides. Tamoxifen added to tumour cells prestimulated with EGF or 17 beta-estradiol failed to abrogate the effect of these compounds on phospholipid turnover. At the same time, treatment of cells with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate fully inhibited the effect of tamoxifen on phospholipid metabolism. The results obtained suggest that the EGF-dependent activation of intracellular phospholipid turnover is under the negative control of progesterone. As for tamoxifen, its effect on cells is independent of EGF and consists, apparently, in the inhibition of protein kinase C activity.

Topics: Adenocarcinoma; Breast Neoplasms; Chromatography, Thin Layer; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

The level of relative accumulation of a 32P radionuclide was measured in 180 tumor samples obtained from 148 patients with stage I--IV breast cancer. Mean initial relative accumulation level (IRAL) was 1477 +/- 174% whereas that following effective conservative treatment (posttreatment) (PRAL)--486 +/- 81% (P < 0.01). The prognostic value of IRAL was established: in a group of stage III breast cancer, those with a high IRAL survived 2 years with no evidence of disease in 55.1 +/- 12.1% whereas those with a low IRAL--in 94.1 +/- 5.7% (P < 0.02). In patients undergoing conservative treatment, low PRAL proved predictive of complete response while high PRAL--of tumor progression. In patients with stage III breast cancer who had responded to preoperative treatment and had had a low PRAL, two-year locoregional recurrence rate was only 13% as compared to 68% in a similar group of patients with high PRAL (P < 0.02).

Topics: Breast Neoplasms; Combined Modality Therapy; Female; Humans; Neoplasm Recurrence, Local; Neoplasm Staging; Phosphorus Radioisotopes; Preoperative Care; Prognosis; Radionuclide Imaging

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Cell Line; Chloramphenicol O-Acetyltransferase; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Ethers, Cyclic; Female; Humans; Isoquinolines; Mammary Tumor Virus, Mouse; Molecular Weight; Okadaic Acid; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Promegestone; Promoter Regions, Genetic; Protein Kinase C; Receptors, Progesterone; Transcription, Genetic; Transfection

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.

Topics: Antibodies, Monoclonal; Autoradiography; Blotting, Northern; Breast Neoplasms; Caseins; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Ethers, Cyclic; Female; Fluorescent Antibody Technique; Gene Expression; Genes, fos; Genes, jun; Humans; Isoenzymes; Kinetics; Marine Toxins; Microcystins; Okadaic Acid; Oligonucleotide Probes; Oxazoles; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Pyrans; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Human breast carcinoma MCF-7 cells seeded on type I collagen-coated dishes were provided with an anchor via the collagen receptor, integrin, and grew as actively as those in plastic tissue culture dishes. In contrast, cells seeded on a layer of soft agar became anchorage-deficient and their growth was significantly inhibited, although the cell viability and the cell cycle distribution were unaffected. Immunoprecipitation analysis revealed that mutant p53 was phosphorylated at tyrosine in the anchorage-provided cells. In contrast, the p53 in the anchorage-deficient cells was present in 2-fold greater amount, but was phosphorylated to a lesser extent. Addition of a potent protein-tyrosine kinase inhibitor, herbimycin A, to the anchorage-provided cells caused an elevated level of p53, and inhibitions of cell proliferation and p53 phosphorylation, without interfering with the cell adhesion to the substratum. These results demonstrated that the growth inhibition by anchorage-deficiency or by herbimycin A is associated with an elevated p53 level and reduced p53 phosphorylation at tyrosine.

Topics: Amino Acids; Antibiotics, Antineoplastic; Autoradiography; Benzoquinones; Breast Neoplasms; Cell Adhesion; Cell Division; Cell Line; Female; Genes, p53; Humans; Lactams, Macrocyclic; Mutation; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Quinones; Rifabutin; Tumor Suppressor Protein p53

Analogues of somatostatin (SS) and luteinizing hormone-releasing hormone (LH-RH) activate tyrosine phosphatases in MIA PaCa-2 human pancreatic cancer cell line membranes and inhibit growth. We compared the substrates phosphorylated by epidermal growth factor (EGF) to those dephosphorylated by the SS analogue RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) and [D-Trp6]LH-RH in cancer cell lines such as MIA PaCa-2 (human pancreatic cancer), HCPC (hamster cheek pouch carcinoma), A-549 (human lung cancer), HT-29 (human colon cancer), and R3230AC (breast cancer). EGF phosphorylated proteins of 170, 65, and 60 kDa and analogues of SS and LH-RH promoted the dephosphorylation of these proteins in MIA PaCa-2 and HCPC cell lines. The EGF receptor is 170 kDa. pp60src (60 kDa) is known to be a substrate for EGF receptor. The LH-RH receptor is also 60 kDa. The effects of RC-160 and [D-Trp6]LH-RH were quantitatively different. Examinations of HT-29, A-549, and R3230AC cancer cell lines revealed no phosphorylation by EGF or dephosphorylation by RC-160 and [D-Trp6]LH-RH. In addition to the 170-, 65-, and 60-kDa proteins, 35-kDa proteins were also phosphorylated in some cancer cell lines. This work demonstrates that analogues of SS and LH-RH can reverse the effects of EGF biochemically as well as functionally.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Autoradiography; Breast Neoplasms; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Kinetics; Lung Neoplasms; Membrane Proteins; Molecular Sequence Data; Pancreatic Neoplasms; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Somatostatin; Triptorelin Pamoate; Tyrosine

The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Base Sequence; Blotting, Northern; Breast Neoplasms; Culture Media; Cysteine; Humans; Immunization; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Phosphoproteins; Phosphorus Radioisotopes; Proto-Oncogene Proteins; Receptor, ErbB-2; Sulfur Radioisotopes; Tumor Cells, Cultured

A method for efficient simultaneous extraction of high-molecular-weight DNA and RNA from solid mammalian tissues including clinical biopsies is described. It is based on the disruption and subsequent melting of deep frozen tissue in the presence of frozen phenol and nucleic acid extraction buffer; this allows for simultaneous disruption of tissue and inactivation of nucleases. The yield is about 0.7-5.8 mg of DNA and 0.5-8.1 mg of total RNA/g of tissue depending upon the tissue type; this is higher than the yield of other methods tested. Analysis of total RNA by denaturing gel electrophoresis, and of DNA and poly(A)+ RNA by Southern and Northern blot hybridization using 32P and biotinylated probes, indicated that c-Ha-ras gene and its transcripts were undegraded. Biotinylated and 32P probes had approximately the same sensitivity in detecting nucleic acids on Southern and Northern blots. This extraction procedure is simple and, when used with biotinylated probes, is rapid, inexpensive, and nonhazardous. The methodology can be modified for use with other clinical samples and cells grown in culture.

Topics: Animals; Biotin; Breast Neoplasms; DNA; Electrophoresis, Agar Gel; Humans; Liver; Molecular Weight; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; RNA

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Enzyme Activation; Epidermal Growth Factor; Female; Humans; Kinetics; Ligands; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Progesterone; Protein Kinases; Receptors, Progesterone

We have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (Mr 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (Mr 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. On molybdate-containing low-salt gradients, PR-6 complexes with 8S B receptors, causing their shift to the bottom of the gradient while A receptors remain at 8 S. Therefore, A and B receptors form separate 8S complexes, and we conclude that A and B do not dimerize in the holoreceptor. Similar gradient studies using salt-containing, molybdate-free buffers show that there are two forms of salt-transformed 4S receptors, comprising either A proteins or B proteins, suggesting that A and B are also not linked to one another in transformed PR. The independence of A- and B-receptor complexes was confirmed by the finding that purified, transformed B receptors bind well to DNA-cellulose. Since PR-6 cross-reacts with nuclear PR, it was used to analyze nuclear PR processing--a down-regulation step associated with receptor loss as measured by hormone binding. Insoluble nuclear receptors and soluble cytosol receptors were measured by immunoblotting following treatment of T47D cells for 5 min to 48 h with either R5020 or progesterone. From 8 to 48 h after R5020 treatment, immunoassayable receptors decreased in nuclei and were not recovered in cytosols. Nuclear receptors also decreased after progesterone treatment but replenished in cytosols between 8 and 24 h after the start of treatment. Thus, processing involves a true loss of nuclear receptor protein, and not just loss of hormone binding activity, and occurs after progesterone or R5020 treatment. This loss is chronic, however, only in R5020-treated cells. Additional studies focused on the covalent modifications of receptors. We previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with [32P]

Topics: Breast Neoplasms; Cell Line; Chromatography, Affinity; Female; Humans; Molecular Weight; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Protein Processing, Post-Translational; Receptors, Progesterone

The bioenergetic state of 15 human tumours was examined with phosphorus-31 magnetic resonance spectroscopy. A striking diversity in metabolic patterns was observed, and significant differences from normal tissue were seen in all cases. A common feature was an elevation of intracellular pH, which may be related to an increase in Na+/H+ exchange during cell activation. It is unlikely that the patterns observed directly correlate with malignancy, but characterisation of the energetic state of a given tumour in a given physiological environment may help in the design and evaluation of interventions for that specific case.

Topics: Adult; Aged; Brain Neoplasms; Breast Neoplasms; Female; Humans; Hydrogen-Ion Concentration; Kinetics; Liver Neoplasms; Magnetic Resonance Spectroscopy; Male; Middle Aged; Neoplasms; Phosphorus Radioisotopes; Spectrum Analysis

We have demonstrated previously the presence of 5-fluorouracil (FUra) residues in L1210 DNA. These findings have been extended to the MCF-7 human breast carcinoma cell line. Cesium sulfate gradient centrifugation has been used to separate the MCF-7 RNA and DNA fractions. Alkali and RNase digests have also been used to remove any possible RNA contaminating the DNA fraction. The purified DNA has been analyzed by high-pressure liquid chromatography following digestion to nucleotides and nucleosides. The results demonstrate that FUra residues are detectable in the DNA of these human breast carcinoma cells following exposure to either FUra of 5-fluorodeoxyuridine. Further, the extend of FUra incorporation in both MCF-7 RNA and DNA is similar with either fluorinated pyrimidine. We also demonstrate that the FUra incorporation in DNA from this human cell line can be enhanced by concurrent incubation with thymidine.

Topics: Breast Neoplasms; Cell Line; DNA Replication; DNA, Neoplasm; Female; Floxuridine; Fluorouracil; Humans; Kinetics; Phosphorus Radioisotopes; Thymidine; Tritium

Radioactive phosphorus effected substantial palliation of intractable bone pain in 17 of 33 (51.5%) women with metastatic carcinoma of the breast and in 14 of 15 (93.3%) men with metastatic carcinoma of the prostate. No significant difference in the overall response rate was found between androgen and parathormone priming prior to radiophosphorus therapy. The degree of response was not dependent on total dose of 32P within the range of 9--18 mCi (333--666 MBq). Myelosuppression was a transient complication in 9 of 33 patients with metastatic breast carcinoma and in 7 of 15 patients with metastatic prostate carcinoma. Symptomatic hypercalcemia was an infrequent complication of radiophosphorus therapy irrespective of the priming regimen.

Topics: Adult; Aged; Bone Neoplasms; Breast Neoplasms; Female; Fluoxymesterone; Humans; Male; Middle Aged; Palliative Care; Parathyroid Hormone; Phosphorus Radioisotopes; Prostatic Neoplasms

The administration of radioactive phosphorus (P-32) for the treatment of pain in metastatic bone lesions from breast cancer shows palliative effects lasting three to nine months. The evolution of the disease is not modified.

Topics: Adult; Aged; Bone Neoplasms; Breast Neoplasms; Female; Humans; Middle Aged; Pain; Palliative Care; Phosphorus Radioisotopes; Testosterone; Time Factors

Eight patients with persistent pain due to disseminated bone metastases from mammary carcinoma were given about 370 MBq (10 mCi) of 32P-pyrophosphate on 10 occasions. All but one of the patients experienced alleviation of pain lasting 1 to 4 months. The side effects, which derived mainly from haematopoetic tissue, prevent the routine use of this compound.

Topics: Blood Cell Count; Bone Neoplasms; Breast Neoplasms; Diphosphates; Female; Humans; Male; Palliative Care; Phosphorus Radioisotopes

Thirty-four patients with cancer of the breast and 12 with cancer of the prostate were treated with testosterone and 32P-sodium phosphate for relief of pain from bony metastases. Thirty were treated with chemotherapy as well, and 34 were treated with external radiation to single ports for localized pain. Of the 46 patients treated, good results were achieved in 34, fair results in six, and no improvement in six. Subsequent marrow depression necessitated transfusion in 10 patients; no other side effect was observed.

Topics: Adult; Aged; Breast Neoplasms; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Phosphorus Radioisotopes; Prostatic Neoplasms

Thirty-four patients with cancer of the breast and 12 with cancer of the prostate were treated with testosterone and 32P-sodium phosphate for relief of pain from bony metastases. Thirty received chemotherapy as well, and 34 received external radiation to single ports for localized pain. Of the 46 patients, 34 had good results, 6 fair, and 6 were failures. Ten patients needed transfusion for marrow depression; no other side effect was observed.

Topics: Adult; Aged; Bone Neoplasms; Breast Neoplasms; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Pain Management; Phosphorus Radioisotopes; Prostatic Neoplasms; Testosterone

Topics: Adult; Bone Neoplasms; Breast Neoplasms; Female; Humans; Male; Neoplasm Metastasis; Pain, Intractable; Palliative Care; Phosphorus Radioisotopes; Prostatic Neoplasms; Testosterone

22 patients suffering from breast neoplasia with particularly painful bone metastasis were treated with radiophosphorus. Only occasionally was an evident recalcification condition encountered and survival, although exceptional in some cases, did not deviate from normal. On the basis, also, of clinical and experimental observations reported in the literature, it is held that the use of 32P in metabolic radiotherapy of bone metastases is worthwhile and is justified because of the encouraging successes obtained, especially in pain remission.

Topics: Adult; Aged; Bone Neoplasms; Breast Neoplasms; Humans; Middle Aged; Neoplasm Metastasis; Phosphorus Radioisotopes; Radiography; Radionuclide Imaging

The results given by the P 32 test and thermography were compared in 92 cases of cancer of the breast, of which 7 appeared after oestrogens or androgens had been administered. After a hormone test of short duration it seemed that reliable results indicating hormone-dependent cancers would only be obtained with the thermographic test.

Topics: Aged; Androgens; Breast Neoplasms; Estrogens; Female; Humans; Middle Aged; Phosphorus Radioisotopes; Thermography

Topics: Blood Platelets; Bone Neoplasms; Breast Neoplasms; Calcium; Female; Follow-Up Studies; Humans; Male; Neoplasm Metastasis; Pain, Intractable; Palliative Care; Parathyroid Hormone; Phosphorus; Phosphorus Radioisotopes; Prostatic Neoplasms; Testosterone

Topics: Breast Neoplasms; Female; Humans; Mammography; Phosphorus Radioisotopes; Radionuclide Imaging; Technology, Radiologic

Topics: Breast Diseases; Breast Neoplasms; Diagnosis, Differential; Evaluation Studies as Topic; Humans; Mammography; Methods; Phosphorus Radioisotopes

Topics: Adult; Aged; Androgens; Body Temperature; Breast Neoplasms; Estrogens; Ethinyl Estradiol; Female; Fluoxymesterone; Humans; Middle Aged; Norethindrone; Phosphorus; Phosphorus Radioisotopes; Skin Temperature; Testosterone; Thermography; Thermometers

Topics: Breast Neoplasms; Female; Humans; Mammography; Mercury Isotopes; Methods; Phosphorus Radioisotopes; Radionuclide Imaging

Topics: Breast Neoplasms; Circadian Rhythm; Female; Humans; Phosphorus Radioisotopes

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Diagnosis, Differential; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Bone and Bones; Bone Neoplasms; Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Bone and Bones; Bone Neoplasms; Breast; Breast Neoplasms; Humans; Nandrolone; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Testosterone

Topics: Adrenalectomy; Breast; Breast Neoplasms; Carcinoma; Female; Humans; Ovary; Phosphorus; Phosphorus Radioisotopes

Topics: Bone and Bones; Bone Neoplasms; Breast; Breast Neoplasms; Humans; Neoplasms; Phosphates; Phosphorus; Phosphorus Radioisotopes

Topics: Animals; Breast Neoplasms; Humans; Mammary Neoplasms, Experimental; Mice; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Exudates and Transudates; Gold Radioisotopes; Humans; Lung Neoplasms; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Radioisotopes

Topics: Bone and Bones; Bone Neoplasms; Breast; Breast Neoplasms; Humans; Male; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Prostatic Neoplasms; Testosterone

Topics: Adrenal Glands; Adrenalectomy; Bone Neoplasms; Breast; Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Pituitary Gland

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cortisone; Humans; Mice; Neoplasm Transplantation; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Bone Marrow Diseases; Bone Neoplasms; Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes

Topics: Breast; Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary; Radioactivity

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary; Radioactivity

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Radioactivity

Topics: Breast Neoplasms; Cervix Uteri; Female; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary; Radioactivity

Topics: Breast; Breast Neoplasms; Female; Humans; Phosphorus; Phosphorus Radioisotopes; Radioactivity; Uterine Neoplasms

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Radiotherapy

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; Radiotherapy

Topics: Breast Neoplasms; Humans; Neoplasms; Neoplasms, Second Primary; Orbit; Orbital Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes

Topics: Animals; Breast; Breast Neoplasms; Humans; Mice; Neoplasms, Experimental; Nucleic Acids; Phosphorus; Phosphorus Radioisotopes; X-Rays

Topics: Bone Neoplasms; Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Skin Neoplasms

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary; Radioactivity

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Breast; Breast Neoplasms; Humans; Neoplasms; Phosphorus; Phosphorus Radioisotopes; X-Rays

Topics: Animals; Breast Neoplasms; Chickens; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred C3H; Neoplasms; Phosphorus; Phosphorus Radioisotopes

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Radioactivity

Topics: Breast Neoplasms; Humans; Phosphorus; Phosphorus Radioisotopes; Radioactivity