Expression of parathyroid hormone-related protein in human inflamed dental pulp.

INTRODUCTION: The aim of this study was to investigate the presence and the role of the parathyroid hormone-related protein (PTH-rP) in the inflamed pulp. MATERIALS AND METHODS: Thirty-four pulp tissue specimens (24 inflamed and 10 normal pulps) from extracted third molars were studied. The presence of PTH-rP was observed by using immunohistochemistry. Negative controls were performed using non immunized rabbit or mouse serum, omitting the primary antibody. RESULTS: The analysis of all the sections of normal pulps showed the presence of PTHrP positive cells only in the odontoblastic zone and in few fibroblasts. Instead all inflamed pulps showed PTHrP positive cells both in vascular zone and in pulp stroma, as well as in the odontoblastic and subodontoblastic zone. CONCLUSION: Several works proved that this peptide plays a role even in angiogenesis process, but its function is controversial. It is possible to hypothesize that PTHrP stimulates angiogenesis, but it is recommended to further conduct research on this area.

Glucocorticoid-binding components in human thymus hyperplasia.

Preliminary experiments on thymocyte suspensions derived from human thymus hyperplasia indicated the presence of specific cytoplasmic receptors binding [3H]-dexamethasone with high affinity and specificity. The receptor was rapidly transferred into the nuclei at 28 degrees but not at 2 degrees. With cell-free preparations and ion-exchange cellulose-impregnated paper filters, thymus cytosol bound [3H]dexamethasone with a dissociation constant of 4.3 x 10(-9) M; the concentration of receptor sites was 9.6 x 10(-14) mole/mg cytosol protein. Cytosol contained binding components that sedimented at approximately 7S and 3.6S (low ionic strength) and at 4S (high ionic strength). Competition studies showed high specificity for glucocorticoids since binding of labeled dexamethasone was inhibited in the presence of 10(-6) M beta-methasone, prednisolone acetate, dexamethasone, corticosterone, cortisol, and cortisone. 17beta-Estradiol, testosterone, and dihydrotestosterone at 10(-6) M did not inhibit specific binding of [3H]dexamethasone. Thus, the dexamethasone-binding components of the human thymus hyperplasia had properties similar to those described for steroid hormone receptors present in target tissues.

Methyl-p-hydroxyphenyllactate-esterase activity in breast cancer: a potentially new prognostic factor in short-term follow-up.

We assayed methyl-p-hydroxyphenyllactate esterase (MeHPLAase) activity in 48 cases of primary breast cancer. MeHPLAase activity did not show significant correlation with estrogen receptor and progesterone receptor levels. No significant relationship was found between enzymatic activity and tumor diameter, lymph node status, mitotic activity, degree of nuclear differentiation, and proportion of the S-phase fraction. During the follow-up period (median, 18.8 months; range, 6-69 months), recurrences were observed in 18 of 48 (37%) cases. The Weibull survival regression model using the enzymatic activity as a continuous covariate showed that levels of enzymatic activity were directly associated with the risk of recurrence (P = 0.02). Assuming the mean value of enzymatic activity as the cutoff value, we found a statistically significant relationship between high MeHPLAase activity and shorter recurrence-free survival. On multivariate analysis, MeHPLAase activity proved to be an independent factor for predicting a short period of recurrence-free survival.

Glucocorticoid receptor determinations in leukemia patients using cytosol and whole-cell assays.

Parallel determinations of glucocorticoid receptors in the cells of patients with various forms of leukemia were made by two assay methods, one using cell-free cytosolic extracts and the other using whole-cell preparations. Both assays revealed saturable binding of triamcinolone acetonide in all cases. The mean equilibrium dissociation constant for the interaction of triamcinolone acetonide with the cytoplasmic receptor at 2 degrees was 9.45 +/- 6.33 (S.D.) nM while that for the whole-cell binding at 37 degrees was 6.13 +/- 3.25 nM, suggesting an increase in receptor affinity at physiological temperatures. Competition experiments with various unlabeled steroids revealed a higher degree of glucocorticoid specificity at 37 degrees in whole-cell suspensions than at 2 degrees in cytosol. In a comparative analysis of 41 leukemic cell specimens, it was found that determinations carried out by whole-cell assay, calculated as number of sites per cell correlated well with those performed by cytosol assay, calculated as fmol/mg protein, independent of the type of leukemia. However, for cells with low receptor content, the two assay methods were more difficult to compare. In agreement with previous reports, the cytosol assay consistently underestimated the number of receptors with respect to the whole-cell assay, particularly in cases of lymphatic leukemia. Furthermore, the underestimation decreased for increasing levels of total cellular receptor. These results suggest that, in addition to possible defects in the cytoplasm-to-nucleus translocation process, the acceptor-binding capacity of the nucleus may also represent one of the factors which determines the levels of assayable cytoplasmic receptors. Moreover, they indicate that the two assay methods furnish nonequivalent information.

Increased cyclooxygenase-2 expression is associated with chemotherapy resistance and poor survival in cervical cancer patients.

PURPOSE: To investigate the expression of cyclooxygenase (COX-2) and its association with clinicopathologic parameters and clinical outcome in patients with cervical cancer. PATIENTS AND METHODS: The study included 84 patients with stage IB to IVA cervical cancer. Patients with early-stage cases (n = 21) underwent radical surgery, whereas patients with locally advanced cervical cancer (LACC) (n = 63) were first administered neoadjuvant cisplatin-based treatment and subjected to surgery in case of response. Immunohistochemical analysis was performed on paraffin-embedded sections with rabbit antiserum against COX-2. RESULTS: COX-2--integrated density values in the overall population ranged from 1.2 to 82.3, with mean plus minus SE values of 27.4 plus minus 2.4. According to the chosen cutoff value, 36 (42.9%) of 84 patients were scored as COX-2 positive. COX-2 levels were shown to be highly associated with tumor susceptibility to neoadjuvant treatment. COX-2 showed a progressive increase from mean plus minus SE values of 19.9 plus minus 8.0 in complete responders through 31.5 plus minus 3.5 in partial responses to 44.8 plus minus 3.9 in patients who were not responsive (P =.0054). When logistic regression was applied, only advanced stage and COX-2 positivity retained independent roles in predicting a poor chance of response to treatment. COX-2--positive patients had a shorter overall survival (OS) rate than COX-2--negative patients. In patients with LACC, the 2-year OS rate was 38% in COX-2--positive versus 85% in COX-2--negative patients (P =.0001). In the multivariate analysis, only advanced stage and COX-2 positivity retained independent negative prognostic roles for OS. CONCLUSION: The assessment of COX-2 status could provide additional information to identify patients with cervical cancer with a poor chance of response to neoadjuvant treatment and unfavorable prognosis.

Cyclooxygenase-1, but not -2, is upregulated in NB4 leukemic cells and human primary promyelocytic blasts during differentiation.

Cyclooxygenase (COX)-1 or -2 and specific prostaglandin (PG) synthases catalyze the formation of various PGs. We investigated the expression and activity of COX-1 and -2 during granulocyte-oriented maturation induced by all-trans-retinoic acid (ATRA) of NB4 cells, originated from a human acute promyelocytic leukemia (APL), and in blasts from APL patients. The expression of COX isoenzymes or prostaglandin synthases was also investigated in circulating granulocytes and human bone marrow. COX-1 was expressed and enzymatically active in NB4 cells and primary blasts. COX-1 mRNA and protein were induced by ATRA. COX-1 protein increased approximately 2-3.5-fold by culture day 3 in NB4 cells and primary blasts, while basal COX-2 expression was very low and unaffected by ATRA. COX-1-dependent PGE(2) biosynthesis increased during differentiation approx. 5-fold. Indomethacin and the selective COX-1 inhibitor SC-560, but not selective COX-2 inhibition, impaired NB4 differentiation, reducing NADPH-oxidase activity, CD11b and CD11c expression. The immunohistochemistry of granulocytes and myeloid precursors in the bone marrow showed a large prevalence of COX-1 as compared to COX-2. In conclusion, COX-1 is induced during ATRA-dependent maturation and appears to contribute to myeloid differentiation both in vitro and ex vivo, and COX-1 activity may potentiate the differentiation of human APL.Leukemia (2004) 18, 1373-1379. doi:10.1038/sj.leu.2403407 Published online 10 June 2004

Tamoxifen modulates the expression of Ki67, apoptosis, and microvessel density in cervical cancer.

PURPOSE: The aim of the study was to investigate if a short-term administration of high-dose Tamoxifen (Tam) could affect the expression of biologically relevant biochemical parameters in cervical cancer tissue. EXPERIMENTAL DESIGN: The study was conducted in 24 patients with histologically confirmed cervical tumors. Biopsies were obtained by colposcopy on day 0 in all patients, who then received either 80 mg/die or 160 mg/die for 5 consecutive days until the second biopsy was obtained. Immunohistochemistry was performed with antiestrogen receptor (ER), anti-Ki67, anticaspase cleavage product of keratin 18 (M30), and anti-CD31 monoclonal antibodies. RESULTS: Eleven (45.8%) of 24 cervical tumors were ER positive. The percentage of Ki67-positive tumor cells in pre-Tam biopsies was significantly higher than the percentage in the corresponding posttreatment biopsies (z = 4.29, P = 0.0001). No difference in the pretreatment percentage of Ki67-positive cells according to ER status was found. The percentage of M30 positivity was higher in post-Tam than in pre-Tam biopsies. Microvessel density values in pre-Tam biopsies were significantly higher than corresponding values in posttreatment tissues (z = -3.72, P = 0.0002). The reduction in the percentage of Ki67-positive tumors was significantly (z = 3.58, P = 0.0003) higher in ER-positive than in ER-negative tumors, whereas no difference in Tam-induced reduction of microvessel density values according to ER status (z = -0.18, P = 0.85) was found. Tam treatment did not induce any change of M30 positivity in ER-positive tumors, whereas in ER-negative tumors, it produced a significant (P = 0.015) increase in the percentage of M30-positive cells in post-Tam versus pre-Tam biopsies. CONCLUSIONS: A short-term treatment with Tam at doses 4-8-fold higher than those in conventional schemes is associated with modifications of biological parameters associated with tumor cell proliferation, apoptosis, and neoangiogenesis in cervical cancer.

The fusion protein MEN 11303 (granulocyte-macrophage colony stimulating factor/erythropoietin) acts as a potent inducer of erythropoiesis.

OBJECTIVE: A fusion protein made of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), referred to as MEN 11303, has been tested for biologic activity using mobilized CD34(+) cells. METHODS AND RESULTS: MEN 11303 and a combination of GM-CSF/EPO produced the same amount of colony-forming unit granulocyte-macrophage (CFU-GM), of burst-forming unit erythroid (BFU-E), and of multipotent CFU-mixed. After 15 days, liquid cultures of CD34(+) cells exposed to MEN 11303 yielded a total cell number larger than that obtained with an equimolar mixture of GM-CSF and EPO, with a clear prevalence of cells exhibiting an erythroid phenotype. A colony-forming cell assay established from CD34(+) cells precultured with MEN 11303 for 7 days yielded a greater amount of BFU-E than GM-CSF/EPO combination. Exposing CD34(+) cells to MEN 11303 for 7 days in liquid culture resulted in higher recoveries of cells expressing a comparatively less differentiated hematopoietic phenotype and of long-term culture initiating cells. A cell-based binding-competition assay using the human EPO-receptor (EPO-R) transfected murine Ba/F3EPOR cell line showed that MEN 11303 bound to EPO-R with a sixfold lower affinity but induced a more sustained receptor phosphorylation. MEN 11303 supported the growth of Ba/F3EPOR cells more efficiently than EPO and remained detectable in the spent culture medium for a longer time. CONCLUSIONS: MEN 11303 and the combination of GM-CSF/EPO are equally potent in recruiting hematopoietic progenitors into cycle, but the fusion protein is superior in promoting the expansion of committed erythroid percursors. Primitive hematopoiesis is less affected by MEN110303 than GM-CSF/EPO combination. Part of these effects may reflect the peculiar interaction of the EPO moiety of MEN 11303 with the EPO-R.

Expression of p15INK4B in normal hematopoiesis.

The cyclin-dependent kinase inhibitor (CDKI) p15INK4B (p15) induces cell cycle arrest in G0/G1 phase. Several studies report deletion or transcriptional loss of the p15 gene in myeloid and lymphoid hematological malignancies, and a possible role as a tumor suppressor gene has been proposed for this CDKI. In this study we evaluated the expression of p15 by cytofluorometric, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) methods in CD34+ progenitors (both during steady state and after chemotherapy and/or granulocyte-colony stimulating factor [G-CSF] administration) and in cells belonging to different hematopoietic differentiative lineages. We found that p15 is not expressed in normal G0/G1-arrested peripheral blood (PB)- or bone marrow (BM)-CD34+ cells. Moreover, p15 is expressed in G0/G1-blocked CD34+ cells mobilized by chemotherapy and G-CSF but not in CD34+ cells mobilized by G-CSF alone. To clarify the role of p15 in normal hematopoiesis, we used flow cytometry to investigate its expression in normal differentiating BM and PB cells. We found that p15 was expressed in cells belonging to the granulocyte-monocyte lineage and in B and T lymphocytes, whereas erythroid and megakaryocytic cells were p15 negative. These findings, which were confirmed both by immunohistochemical and RT-PCR analysis, definitely establish a linkage between p15 expression and granulocyte-monocyte differentiation.

Tamoxifen and quercetin interact with type II estrogen binding sites and inhibit the growth of human melanoma cells.

The mechanism of the antiproliferative activity of tamoxifen on melanoma cells in vitro and in vivo is poorly understood, as it is not mediated by the antiestrogenic properties of tamoxifen. Using a whole-cell assay and nuclear and cytosolic radio-binding experiments with [3H]-estradiol as tracer, we found that MNT1, M10, and M14 melanoma cell lines as well as primary tumors expressed type II estrogen binding sites that bind tamoxifen and the flavonoid quercetin with similar affinity (KD 10-25 nM). Cell count and clonogenic assay showed both compounds to inhibit melanoma cell growth in a concentration-dependent manner in the range of concentrations between 1 nM and 1 microM. Neither the pure antiestrogen ICI-182780 nor the 3-rhamnosylglucoside of quercetin, rutin, bound to type II estrogen binding sites or inhibited cell growth. Our results suggesting that tamoxifen and quercetin can inhibit melanoma cell growth by interacting with type II estrogen binding sites help explain the reported effectiveness of tamoxifen, particularly in estrogen-receptor-negative tumors, and stress the potential role of quercetin in the treatment of melanoma.

Type II estrogen-binding sites and 17 beta-hydroxysteroid dehydrogenase activity in human peripheral blood mononuclear cells.

Type II estrogen-binding sites (type II EBS) have been demonstrated in human peripheral blood mononuclear cells (PBMC) using a whole cell assay with [6,7-3H]estradiol [( 3H]E2) as tracer. During whole cell incubations for 60 min at 37 C for type II EBS quantification, we found that PBMC contain 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity, which led to errors in estimating type II EBS concentrations by diminishing, by about 70%, the amount of available labeled E2. On the other hand, after 150 min at 4 C only 16% of the tracer was converted to estrone. Thus, we measured the maximal steady state binding in PBMC by incubating the cells with [3H]E2 at 4 C for 150 min. Equilibrium binding analysis of PBMC yielded sigmoid saturation curves with a saturation point at a ligand concentration of about 40 nmol/L. Scatchard analysis of binding data yielded a concave plot, which together with a Hill coefficient of 2.13, suggests that the type II EBS may have multiple binding sites which display positive cooperativity. The apparent equilibrium dissociation constant (Kd), determined from the [3H]E2 concentration required for half-saturation, was about 22 nmol/L. The type II EBS were estrogen specific, as demonstrated by competition experiments. Only those steroids with estrogenic activity inhibited binding of [3H]E2; nonestrogenic steroids did not. The type II EBS were found to be 3S macromolecules based on analysis of postlabeled fractions prepared by sucrose density gradient centrifugation. The number of type II EBS in PBMC from normal women was highest during the late follicular-early luteal phase of the menstrual cycle. We conclude that human PBMC specifically take up, retain, and metabolize E2.

Beta-carotene antagonizes the effects of eicosapentaenoic acid on cell growth and lipid peroxidation in WiDr adenocarcinoma cells.

The effects of combinations between eicosapentaenoic acid (EPA) and beta-carotene on cell growth and lipid peroxidation were investigated in human WiDr colon adenocarcinoma cells. EPA alone was able to inhibit the growth of WiDr cells in a dose- and time-dependent manner. Such an inhibition involved fatty acid peroxidation, as shown by the remarkable increase in the levels of Malondialdehyde (MDA) in EPA-treated cells. Beta-carotene was capable of reducing the growth inhibitory effects of EPA and the levels of MDA in a dose- and a time-dependent manner. In addition, EPA increased beta-carotene consumption in WiDr cells. This study provides evidence that beta-carotene can antagonize the effects of EPA on colon cancer cell growth and lipid peroxidation.

beta-carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells.

This is the first report demonstrating a relationship between apoptosis induction and changes of intracellular redox potential in the growth-inhibitory effects of high concentrations of beta-carotene in a tumor cell line. beta-Carotene inhibited the growth of human WiDr colon adenocarcinoma cells in a dose- and time-dependent manner, induced apoptosis, and blocked Bcl-2 expression. These effects were accompanied by an enhanced production of intracellular reactive oxygen species (ROS). The addition of the antioxidant alpha-tocopherol blocked both the pro-oxidant and the growth-inhibitory effects of the carotenoid. These findings suggest that beta-carotene may act as an inductor of apoptosis by its pro-oxidant properties.

Synergistic antiproliferative activity of tamoxifen and cisplatin on primary ovarian tumours.

We looked for the presence of the so-called type II oestrogen binding sites (EBS), in four oestrogen (ER) and progesterone (PR) receptor negative primary ovarian tumours. Moreover, the colony-forming assay was used to evaluate the response of ovarian cancer cells from these primary tumours to tamoxifen and cisplatin used alone or in combination. All tumours contained type II EBS, and tamoxifen was able to compete for [3H] oestradiol binding to these sites. Cisplatin and tamoxifen exhibited a dose-dependent inhibition of colony formation in a range of concentrations between 10 and 1000 micrograms/l and 37 and 3710 micrograms/l, respectively. The combination of the two drugs resulted in a synergistic antiproliferative activity, with a potentiation up to and beyond 50-fold. Our results show that in ovarian cancer tamoxifen interacts with type II EBS with an affinity consistent with the concentration effective both in inhibition of colony formation and in synergising cisplatin activity. Based on the experiments performed the action of tamoxifen on cell growth is independent of ER expression, and could be mediated by type II EBS. The possibility that the association of tamoxifen and cisplatin may result in an improved clinical response in ovarian cancer should be investigated.

Antiproliferative effect of silybin on gynaecological malignancies: synergism with cisplatin and doxorubicin.

The aim of this study was to test the antiproliferative activity of silybin, a flavonoid, on human ovarian and breast cancer cell lines. Since flavonoids are thought to act through Type II oestrogen binding sites (Type II EBS), silybin binding to Type II EBS was also examined. Silybin, used in concentrations from 0.1 to 20 microM, exerted a dose-dependent growth inhibitory effect on OVCA 433, A2780 parental and drug-resistant ovarian cancer cells, and MCF-7 doxorubicin (DOX)-resistant breast cancer cells (IC50 = 4.8-24 microM). Both L and D diastereoisomers of silybin were effective in inhibiting A2780 WT cell growth (IC50 = 14 and 20 microM, respectively). Flow cytometry revealed that silybin decreased the percentage of cells in the S and G2-M phases of the cell cycle with a concomitant increase in cells in the G0-G1 phase. Silybin was able to compete with [3H]E2 for nuclear but not cytosolic Type II EBS. Its affinity parallels its efficacy in inhibiting cell proliferation. Furthermore, silybin (0.1 and 1 microM) potentiates the effect of cisplatin (CDDP) (0.1-1 micrograms/ml) in inhibiting A2780 WT and CDDP-resistant cell growth. Similar results were obtained on MCF-7 DOX-resistant cells when silybin (0.1 microM) was associated with doxorubicin (0.1-10 micrograms/ml). As assessed by the Berembaum isobole method, the effect of silybin-CDDP and silybin-DOX combinations results in a synergistic action. Using the 'stem cell assay' described by Hamburger and Salmon [Science 1977, 197, 461-463], we found that silybin exerted a dose-dependent inhibition of clonogenic efficiency of cells derived from three ovarian tumours (IC50 = 7.4, 4 and 6.4 microM, respectively). Since CDDP and DOX are the two most commonly used drugs for gynaecological tumours, the clinical application of silybin is currently under investigation in our institute.

Cyclooxygenase-2 (COX-2) expression in locally advanced cervical cancer patients undergoing chemoradiation plus surgery.

PURPOSE: To investigate whether cyclooxygenase-2 (COX-2) could be a marker of clinical outcome in cervical cancer patients undergoing concomitant chemoradiation plus surgery. METHODS AND MATERIALS: The study included 33 locally advanced cervical cancer patients; all underwent neoadjuvant chemoradiation, and responsive patients underwent radical surgery. Immunohistochemistry was performed with rabbit antiserum against COX-2. RESULTS: COX-2 integrated density values (IDVs) in the tumor component ranged from 1.4 to 72.3 (median 15.0); in stromal inflammatory cells, COX-2 IDVs ranged from 1.4 to 96.0 (median 16.0). A statistically significant inverse relation was found between the COX-2 IDVs of the tumor vs. the stromal inflammatory component (r = -0.52, p = 0.0017). When the ratio between COX-2 IDV in the tumor vs. the stromal compartment was 1) tumor/stroma COX-2 IDV ratio. Patients with a high tumor/stroma COX-2 IDV ratio had a shorter disease-free survival than did those with a low tumor/stroma COX-2 IDV ratio (p = 0.030). Similarly, those with a high tumor/stroma COX-2 IDV ratio had a shorter overall survival (p = 0.033). CONCLUSION: The assessment of COX-2 status in both the tumor and the stromal compartment could provide additional information in the prognostic characterization of cervical cancer patients administered concomitant chemoradiation plus surgery.

Detection of growth hormone-producing cells in human thymus by immunohistochemistry and non-radioactive in situ hybridization.

It is well recognized that growth hormone (GH) may act as a growth and differentiation factor for the thymus gland. Recently, it has been reported that Pit-1/GHF-1 transcription factor, which controls the expression of both GH and prolactin, is expressed in stromal (not lymphoid) cells of human thymus. Here, we demonstrated by immunohistochemistry and in situ hybridization the presence of distinct GH-producing epithelial cell subsets in human thymus. The cells positive for GH mRNA and GH-immunoreactive substance are both located in the same thymus compartments, i.e., along the thymus capsule, in the subcapsular cortex, and within the septa. Local concentration of GH higher than systemic ones, in combination with other factors, may be important in regulating the thymic microenvironment necessary for T-lymphocyte differentiation.

Detection of synaptophysin-producing cells in human thymus by immunohistochemistry and nonradioactive in situ hybridization.

We investigated human thymic tissue by immunohistochemistry and in situ hybridization for the presence of synaptophysin-producing cells. Our results indicate that anti-synaptophysin antibody detected immunoreactive material in nerve fibers around vessels located in major thymic septa, in a relevant number of cortical epithelial cells, and in scattered epithelial cells in the medulla. The epithelial nature of synaptophysin-positive cells was documented by the co-expression of cytokeratins as revealed by double immunofluorescence. In situ hybridization studies revealed the presence of synaptophysin mRNA in cells mainly located in the cortex, the specific fluorescent signals being localized in the cell cytoplasm. Western blot analysis using an affinity-purified polyclonal antibody revealed an immunoreactive band of about 38 kD in the extracts from unfractionated thymic tissue and from epithelial cell-enriched fractions. No staining was observed in isolated thymocytes. The expression of synaptophysin in epithelial cells of the thymic cortex suggests that this protein may be involved in secretory activities related to T-cell maturation.

Inherited macrothrombocytopenia with distinctive platelet ultrastructural and functional features.

We report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65 x 10(9)/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder. Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were approximately 2 standard deviations above control values, in spite of a reduced maximal biosynthetic capacity of thromboxane from giant platelets assessed in vitro during whole blood clotting. This inherited platelet disorder shows structural and functional features which allow to distinguish it from other syndromes associated with giant platelets. We also propose to include ultrastructural and cytoskeletal studies in the diagnosis as well as in the classification of inherited giant platelet disorders.

Interaction of tamoxifen with cytosolic and nuclear type II estrogen binding sites (type II EBS).

The aim of this study was to investigate the interaction of tamoxifen (TAM) with the so-called Type II estrogen binding sites (Type II EBS) in both the cytosolic and the nuclear fraction of the ER-negative A 2780 human ovarian cancer cell line and in an ER-negative ovarian cancer tissue. Although cytosolic and nuclear Type II EBS in A 2780 cells showed substantially similar binding characteristics in terms of ligand affinity and specificity, TAM, while exhibiting the ability to displace [3H]estradiol from cytosolic Type II EBS failed to interact with nuclear Type II EBS. The ability of TAM to interact only with cytosolic Type II EBS seems also to be a characteristic of ovarian cancer tissue and to be shared by several TAM metabolites. The hypothesis that the interaction of TAM with cytosolic Type II EBS could mobilize the true endogenous ligand of Type II EBS which would become available for binding to nuclear Type II EBS was tested by incubating the nuclear fraction with the cytosolic fraction. In the presence of cytosol, TAM acquires the ability to displace the tracer from nuclear Type II EBS but when the cytosolic fraction was DCC, stripped in order to remove the endogenous ligand, the competing activity of TAM for nuclear Type II EBS was abolished. Our results suggest that TAM does not interact with nuclear Type II EBS, but can favor the nuclear binding of endogenous ligand by displacing it from cytosolic Type II EBS.