Estradiol receptor levels in human breast carcinomas.

The presence or absence of a specific estradiol-binding protein receptor in the cytoplasm of primary and secondary tumour cells has been used by physicians as an important guide in deciding whether to use hormonal therapy for patients with metastatic breast cancer. This report gives the levels of estradiol receptors in the cytosol of 228 primary and secondary breast tumours, measured by a sensitive multiple-point assay in which dextran-coated charcoal separated bound form unbound estrogen. The data were analysed with a Scatchard plot. Of the 175 primary and 53 secondary tumours 53% and 32% respectively gave positive results. The mean receptor level in the primary tumours was significantly higher among older patients and increased with age. With metastatic lesions positive results were more common in lymph node samples tha in skin nodule samples.     

Study of the binding of iron ions by melanoprotein granules of the eye pigment epithelium using gamma-resonance spectroscopy

Interaction between Fe2+ ions and melanoproteid granules (MPG) of bovine eye pigment epithelium was studied by gammaresonance spectroscopy. MPG was found to form complexes with bi- and three-valent ferrum ions. MPG can both directly bind Fe2+ ions and oxidize them Fe3+ inactive in prooxidant state and bind the latter. The activity of ferrum ion binding increases when suspension is illuminated with visible light and pH of the incubation solution increases. The protein part of MPG does not participate in the complex formation with ferrum ions. The complex formation proceeds mainly by carboxyl, amino- and imino-groups of melanin polymer.     

Hormone-dependence of experimental mammary tumours

The effect of hormones on mammary tumours induced experimentally in animals are revised. It is stated that the effect on tumoural induction and/or growth may depend on their concentration and on the moment, in relation to animal exposure to the carcinogen, in which they are administered. The presently known action mechanisms are also indicated, bringing out the importance of the increased level of differentiation which high estrogen doses induce, as well as some effects of progesterone and pregnancy in the protection against experimental cancer and its promoters. The efficacy of therapeutic ablative operations--ovariectomy, hypophysectomy and adrenalectomy--and additive ones--antihormones--in tumor regression, is shown. Finally the molecular bases of hormonedependence of experimental mammary tumours is established, while trying to provide an integrated concept between: hormones, receptor, growth factors, oncogens and experimental carcinogenesis.     

Subcellular localisation of VEGF in different pituitary cells. Changes of its expression in oestrogen induced prolactinomas

Summary Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.     

E-cadherin和CD44V6在食管上皮癌变过程及癌组织中的表达

研究不同类型的食管上皮增生和癌组织的 E- cadherin (E- cad)和 CD44 V6的表达 ,并探讨其与食管癌发生和发展的关系。应用免疫组织化学 SABC法 ,观察 10例正常、 3例消化性溃疡、 2 5例单纯性增生、 15例不典型增生的食管粘膜上皮 ,5例食管原位癌与 5 4例浸润癌组织中的 E- cad和 CD44 V6蛋白的表达情况。结果显示正常食管鳞状上皮和高分化肿瘤细胞膜和细胞浆 E- cad和 CD44 V6染色 ,非典型增生、低分化肿瘤细胞两种蛋白抗体表达减弱或呈阴性。E- cad和 CD44 V6的表达与癌组织的组织学分级、类型和淋巴结转移有关 (P<0 .0 1,P<0 .0 5 ) ,与癌组织的浸润深度无关 (P>0 .0 5 )。提示E- cad和 CD44 V6表达减弱是癌组织低分化和高度恶性的生物学标志 ,但其与淋巴结转移的关系有待进一步研究     

Strongly Magnetic Iron Nanoparticles Improve the Diagnosis of Small Tumours in the Reticuloendothelial System by Magnetic Resonance Imaging

Despite advances in non-invasive medical imaging, accurate nodal staging of malignancy continues to rely on surgery. Superparamagnetic iron oxide nanoparticles (IONP) with lymphotropic qualities have shown some promise as contrast agents for MRI of the lymph nodes, but recent large-scale studies failed to show consistent detection of tumours below 5 mm. Herein we compare imaging of splenic and lymph node tissue using iron/iron oxide core/shell nanoparticles (Fe NP) that have superior magnetic qualities to IONP, to determine whether improved negative contrast in T₂-weighted MRI can enhance the diagnosis of small tumours in the reticuloendothelial system. To provide an in vivo pre-clinical model of human lymph node micrometastases, breast cancer cells were injected into the spleens of mice, providing localised areas of tumour growth. MR images of groups of tumour-bearing and sham-treated animals were generated using a 1.5 T imaging system and analysed by two independent, blinded radiologists. Fe NP improved the sensitivity and specificity of MRI when compared to IONP, enabling accurate detection of tumours as small as 1–3 mm. The use of Fe NP as contrast agents have the potential to improve the diagnostic accuracy of MRI in cancer patients, leading to more rapid and effective treatment.     

17β-Hydroxysteroid dehydrogenase type 14 is a predictive marker for tamoxifen response in oestrogen receptor positive breast cancer

Introduction

17β-hydroxysteroid dehydrogenases (17βHSDs) are important enzymes regulating the pool of bioactive steroids in the breast. The current study was undertaken in order to evaluate implications of 17βHSD14 in breast cancer, measuring 17βHSD14 protein expression in breast tumours.

Methods

An antibody targeting the 17βHSD14 antigen was generated and validated using HSD17B14-transfected cells and a peptide-neutralising assay. Tissue microarrays with tumours from 912 post-menopausal women diagnosed with lymph node-negative breast cancer, and randomised to adjuvant tamoxifen or no endocrine treatment, were analysed for 17βHSD14 protein expression with immunohistochemistry.

Results

Results were obtained from 847 tumours. Patients with oestrogen positive tumours with high 17βHSD14 expression had fewer local recurrences when treated with tamoxifen (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) compared to patients with lower tumoural 17βHSD14 expression, for whom tamoxifen did not reduce the number of local recurrences (HR 1.19; 95% C.I. 0.54–2.59; p = 0.66). No prognostic importance of 17βHSD14 was seen for systemically untreated patients.

Conclusions

Using a highly specific validated antibody for immunohistochemical analysis of a large number of breast tumours, we have shown that tumoural expression levels of 17βHSD14 can predict the outcome of adjuvant tamoxifen treatment in terms of local recurrence-free survival in patients with lymph node-negative ER+ breast cancer. The results need be verified to confirm any clinical relevance.     

Diamine and polyamine oxidase activities in normal and transformed cells

1. The activity of diamine oxidase (EC 1.4.3.6) in normal rat kidney cells and in normal rat kidney cells transformed by avian sarcoma virus (B77 strain) growing in tissue culture varies with the stage of growth. There is an initial stimulation of activity by 24h after seeding, followed by a steep decline during exponential growth (48-72h). Enzyme activity decreases even further as the cells reach saturation density (confluence) after 4 days in culture when the activity in normal rat kidney cells is twice as high as that in transformed cells. 2. Differences of about the same order of magnitude are observed between transformed human cells HeLa, HEp2 (a human epithelioid carcinoma) and normal human fibroblasts, in chicken cells between normal myeloblasts and leukaemic myeloblasts, and in rats between biopsy material from normal mammary tissue and 9,10-dimethylbenz[a]anthracene-induced mammary tumours. 3. Polyamine oxidase activity also varies with the growth of transformed rat kidney cells, but shows no significant variation with the growth of normal rat kidney cells between 24 and 96h after seeding. The activity in cells at confluence is from 3- to 5-fold lower in the transformed than in the normal rat kidney cells. 4. A similar 5-10-fold decrease in activity has been found in 9,10-dimethylbenz[a]anthracene-induced mammary tumours in rats and in human oesophageal tumours. 5. Possible reasons for these observations and the contribution of these two enzymes to cellular putrescine concentrations are discussed.     

Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein.

Bispecific antibodies directed against tumour associated antigens and the T cell receptor component CD3 for recruitment and tumour targeted activation of T cells represent a novel class of highly specific immunotherapeutics for cancer. We here describe the construction, eukaryotic expression and in vitro functional activity of a new T cell activating bispecific reagent, termed TTS for T cell targeting to the tumour stroma, comprised of a CD3 specific single chain antibody derivative (scFv) fused C-terminally to a 'fibroblast activation protein' (FAP) specific scFv that targets cytotoxic effector cells to FAP. FAP is highly expressed in the vascularised tumoural stroma of most lung, breast and colon carcinomas. It thus represents a selectively tumour associated, yet common marker of many solid tumours and is a potentially ideal candidate marker for efficient targeting of immune effector cells.     

NuMA overexpression in epithelial ovarian cancer

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon.NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis.Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008).NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.     

Thyroid hormones and the level of divalent iron ions in the rabbit liver

Experimental hypothyreosis in animals was obtained by means of extirpation of thyroid gland; hyperthyreosis was obtained by thyroxine injections intraperitoneally for 10 days. Content of Fe2+ ions of the free pool (not bound with ferritine) was estimated with the ESR method, after treatment with sodium nitrite. A decrease in Fe2+ ions content is observed in liver homogenate of hypothyroid rabbits, and an increase is observed in that of the hyperthyroid animals. Content of Fe2+ ions in cells of liver tissues correlates with the oxidation capacity of the mitochondrial lipids. The changes in the rate of lipid peroxidation under thyroid pathology is supposed to result from changes in free ferrum content.     

Manifestations of adaptive processes in the epithelium of the renal glomerular filter

Fifteen kidney biopsy samples+, obtained from children suffering from the primary nephrotic syndrome, characterized with a high selective proteinuria and non-inflammatory character of changes in the renal corpuscles have been investigated after hormonotherapy (11 cases before and 4 cases 2-3 weeks after it). In podocytes certain disturbances in the lysosomal-vascular apparatus develop with a successive appearance of degeneration, desquamation and even necrosis of these cells. After prednisolone++ treatment edema of cytoplasm of the cells and their processes disappears, amount and size of the filtration slits are partly restored.     

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.     

Rapid melanization of bomirski amelanotic melanoma cells in cell culture

Transfer of Bomirski amelanotic melanoma cells from in vivo to in vitro growth conditions results in occurrence of rapid melanization in their cytoplasm. The melanized cells from primary cell culture initiate tumours in hamsters, which do not contain traces of melanin and resemble typical amelanotic melanoma.     

Preliminary study on the distribution of selected elements in cancerous and non-cancerous kidney tissues

In this study special interest was given to trace elements recognized as to be carcinogenic to humans. The kidney tissue sections were analyzed in order to determine the concentrations of elements present in the sample. The Synchrotron Radiation Induced X-ray Emission (SRIXE) technique was applied using a white photon microbeam. The results from cancerous parts of the kidney tissues were compared to non-cancerous parts and to the control group. In addition the iron concentration level was determined in the serum of those patients. Two-dimensional scans are presented to illustrate the differences between perfused and not-perfused tissues. According to this study there is no significant difference in the Mn concentration between cancerous and non-cancerous parts of the kidney, but the concentrations of Cd, Cr, Ti, V, Cu, Se, and Zn are at a lower concentration level in the cancerous parts than in the non-cancerous parts. A converse observation has been made for Fe. This may be associated with different metabolism and dynamics of the cancer process and both higher vascularization and need of higher blood supply in the cancerous tissue. The two-dimensional scanning of thin kidney sections showed differences in the trace element distributions depending on the analyzed samples: perfused and non-perfused. Perfusion removed blood mostly from the peritubular capillaries while in the glomerulus some capillaries had a relatively high Fe content. A low Fe concentration was observed in nephron tubules while a converse observation has been made for Cd. This may indicate that Cd is localized in the cells but not in the blood.     

Lectin Agglutinability of Mammary Tumours with Differing Metastatic Colonisation Potentials

A new fully quantitative method for assessment of lectin agglutinability has been used in this investigation to compare the surface composition of cells from tumours with high and low pulmonary colonisation potential. As in our previous work, we have used only primary (i.e., naturally-occurring) mammary tumours in mice. It was found that agglutinability with the lectins Concanavalin-A and Wheatgerm agglutinin bore no relationship to the pulmonary colonisation potential of the primary mammary tumour. However, cells from disaggregated secondary deposits of tumours which manifested high colonisation potential were consistently less agglutinable than the cells of the primary tumours from which they were derived.     

Neuron specific enolase-positive breast carcinomas

Ninety-eight patients treated for breast carcinomas were followed from 54 to 75 months after primary diagnosis. All had undergone a modified radical mastectomy with removal of axillary lymph nodes. 36 breast carcinomas were NSE-positive and 62 were negative. NSE-positive tumours were significantly more frequently estrogen receptor-positive than the NSE-negative tumours, and the estrogen receptor values were higher in the NSE-positive groups. Patients with NSE-positive tumours and patients with NSE-negative tumours did not differ with regard to presence of lymph node metastases at the time of primary surgery. However, the study showed that patients with NSE-positive tumours had a tendency towards more lymph node metastases after primary surgical intervention, but a better outcome than patients with NSE-negative tumours and metastases. This study, with a 5-year follow up, failed to demonstrate any major prognostic significance of immunostaining for NSE.     

Effect of N-stearoylethanolamine on the DNA fragmentation intensity in tumour and extratumoral tissues of the human adrenal cortex

The effect of different concentrations of N-stearoylethanolamine (NSE 18:0) on fragmentation of DNA in the tumoural and extratumour tissues of the adrenal glands in vitro was studied. In this work the following types of tissue were investigated: extratumoural tissue from patients with hormonally active tumours, benign tumour tissue (hormonally active and hormonally inactive), tissue of malignant tumours and hyperplasic tissue of the adrenal glands (Itsenko-Cushing disease). It has been established that the NSE increases the intensity of DNA fragmentation only in the tissue of hormonally inactive tumours. Benign hormonally active tumours, malignant tumours and hyperplastic tissue of the adrenal glands were resistant to the NSE. The possible mechanisms of resistance to the drug are discussed.     

The immunohistochemical expression of stress-response protein (srp) 60 in human brain tumours: relationship of srp 60 to the other five srps, proliferating cell nuclear antigen and p53 protein

This study analyzed the expression of stress-response (heat-shock) protein 60 (srp 60) in a series of 158 human brain tumours. Immunohistochemical procedures were employed; cells of the human cervical cancer line HeLa S3 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm. Approximately half of the glioblastomas multiforme (17/31), breast carcinoma metastases (6/10), and lung carcinoma metastases (5/11) as well as about one-third of the astrocytomas (5/13) and meningiomas (8/23) had tumour cells that expressed srp 60. A positive reaction for srp 60 was also seen in some medulloblastomas (2/16), primitive neuroectodermal tumours (PNETs) (2/11), schwannomas (2/21), and pituitary adenomas (2/7), but no positive reactions were observed with oligodendrogliomas and ependymomas. Compared with srp 60-negative tumours, srp 60-positive tumours coexpressed one or more stress-related proteins, among which srp 90, srp 72, srp 27, alphaB-crystallin and ubiquitin occurred with higher frequencies; a high correlation between srp 60 and the other five srps (0.88 - 0.97, p<0.01, Pearson correlation coefficient) was observed in srp 60-positive tumours. In contrast, the correlation coefficient in srp 60-negative tumours was not significant (-0.26 - 0.71). There was a tendency for the proliferating cell nuclear antigen (PCNA)-labeling index to be higher in glioblastomas, astrocytomas, medulloblastomas, PNETs, and breast and lung carcinoma metastases that expressed srp 60 than in those that did not. No significant immunohistochemical reactions of srp 60, PCNA and p53 protein were seen with sections of normal brain tissues. We conclude that primary and metastatic tumours of the brain produce srp 60 and that srp 60 in certain brain tumour cells may coexpress the other five srps. In addition, srp 60 expression might depend, in part, on proliferating potential.     

Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.