MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion.

BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

Incidence of leukaemia and other malignant diseases following injections of the short-lived alpha-emitter 224Ra into man.

We performed an epidemiological study on 1,471 ankylosing spondylitis patients treated with repeated intravenous injections of the short lived alpha-emitter (224)Ra (excluding radiation therapy with X-rays) between 1948 and 1975. These patients have been followed together with a control group of 1,324 ankylosing spondylitis patients treated neither with radioactive drugs nor with X-rays. The mean follow-up time was 26.3 years in the exposed and 24.6 years in the control group. To date, causes of death have been ascertained for 1,006 exposed patients and 1,072 controls. Special emphasis was placed on the reporting of malignant diseases. Expected numbers of cases were computed for the age, sex and calendar year distribution of both groups using cancer registry incidence rates. In the exposed group 18 cases of kidney cancer (vs. 9.1 cases expected, P < 0.01) and 4 malignant thyroid tumours (vs. 1.2 cases expected, P = 0.03) were observed. In the control group the observed cases for these tumours were not significantly elevated. The most striking observation, however, were the 21 cases of leukaemia in the exposed group (vs. 6.8 cases expected, P < 0.001) compared to 12 cases of leukaemia in the control group (vs. 7.5 cases expected). Further sub-classification of the leukaemias demonstrated a high increase of myeloid leukaemia in the exposed group (12 cases observed vs. 2.9 cases expected, P < 0.001), and out of these, especially a high excess of acute myeloid leukaemias (7 cases observed vs. 1.8 expected, P = 0.003). In the controls the observed cases are within the expected range (4 myeloid leukaemias vs. 3.1 cases). This increase in total leukaemias as well as particularly in myeloid leukaemias is significant in direct comparison between the exposed and control groups too (P < 0.05). The enhanced leukaemia incidence in the exposed group is in line with the observation of increased leukaemia incidence in mice injected with (224)Ra.

Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

Progress in updating the European Radiobiology Archives.

PURPOSE: The European Radiobiology Archives (ERA), together with corresponding Japanese and American databases, hold data from nearly all experimental animal radiation biology studies carried out between 1960 and 1998, involving more than 300,000 animals. The Federal Office for Radiation Protection, together with the University of Cambridge have undertaken to transfer the existing ERA archive to a web-based database to maximize its usefulness to the scientific community and bring data coding and structure of this legacy database into congruence with currently accepted semantic standards for anatomy and pathology. METHODS: The accuracy of the primary data input was assessed and improved. The original rodent pathology nomenclature was recoded to replace the local 'DIS-ROD' (Disease Rodent) formalism with Mouse Pathology (MPATH) and Mouse Anatomy (MA) ontology terms. A pathology panel sampled histopathological slide material and compared the original diagnoses with currently accepted diagnostic criteria. RESULTS: The overall non-systematic error rate varied among the studies between 0.26% and 4.41%, the mean error being 1.71%. The errors found have been corrected and the studies thus controlled have been annotated. The majority of the original pathology terms have been successfully translated into a combination of MPATH and MA ontology terms. CONCLUSIONS: ERA has the potential of becoming a world-wide radiobiological research tool for numerous applications, such as the re-analysis of existing data with new approaches in the light of new hypotheses and techniques, and using the database as an information resource for planning future animal studies. When the database is opened for new data it may be possible to offer long-term storage of data from recent and future animal studies.

Impact of Inter-Individual Variance in the Expression of a Radiation-Responsive Gene Panel Used for Triage.

In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.

Linking DNA damage to medulloblastoma tumorigenesis in patched heterozygous knockout mice.

Hemizygous Ptc1 mice have many features of Gorlin syndrome, including predisposition to medulloblastoma development. Ionizing radiation synergize with Ptc1 mutation to induce medulloblastoma only in neonatally exposed mice. To explore the mechanisms underlying age-dependent susceptibility, we irradiated Ptc(neo67/+) mice at postnatal day 1 (P1) or 10 (P10). We observed a dramatic difference in medulloblastoma incidence, which ranged from 81% in the cerebellum irradiated at P1 to 3% in the cerebellum irradiated at P10. A striking difference was also detected in the frequency of cerebellar preneoplastic lesions (100 versus 14%). Our data also show significantly lower induction of apoptosis in the cerebellum of medulloblastoma-susceptible (P1) compared to -resistant (P10) mice, strongly suggesting that medulloblastoma formation in Ptc1 mutants may be associated with resistance to radiation-induced cell killing. Furthermore, in marked contrast with P10 mice, cerebellum at P1 displays substantially increased activation of the cell survival-promoting Akt/Pkb protein, and markedly decreased p53 levels in response to radiation-induced genotoxic stress. Overall, these results show that developing cerebellar granule neuron precursors' (CGNPs) radiosensitivity to radiation-induced cell death increases with progressing development and inversely correlates with their ability to neoplastically transform.

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease.

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.

The Rb1 tumour suppressor gene modifies telomeric chromatin architecture by regulating TERRA expression.

The tumour suppressor gene (Rb1) is necessary for the maintenance of telomere integrity in osteoblastic cells. We now show that the compaction of telomeric chromatin and the appropriate histone modifications of telomeric DNA are both dependent upon Rb1-mediated transcription of the telomere-derived long non-coding RNA TERRA. Expression of TERRA was reduced in Rb1 haploinsufficient cells, and further decreased by shRNA-mediated reduction of residual Rb1 expression. Restoration of Rb1 levels through lentiviral transduction was sufficient to reestablish both transcription of TERRA and condensation of telomeric chromatin. The human chromosome 15q TERRA promoter contains predicted retinoblastoma control elements, and was able to confer Rb1-dependent transcription upon a promoterless reporter gene. Chromatin immunoprecipitation revealed preferential binding of phosphorylated over non-phosphorylated Rb1 at the TERRA promoter. As Rb1-deficient cells show increased genomic instability we suggest that this novel non-canonical action of Rb1 may contribute to the tumour suppressive actions of Rb1.

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

E-cadherin gene mutations provide clues to diffuse type gastric carcinomas.

The calcium-dependent homophilic cell adhesion molecule and candidate suppressor gene, E (epithelial)-cadherin, plays a major role in the organization and integrity of most epithelial tissues. Diffusely growing gastric carcinomas show markedly reduced homophilic cell-to-cell interactions. We speculated that mutations in the E-cadherin gene may be responsible for the scattered phenotype of this type of carcinoma. For that reason we have examined E-cadherin in 26 diffuse type, 20 intestinal type and 7 mixed gastric carcinomas (Laurén's classification) at the DNA, RNA, and protein levels. Reverse transcription polymerase chain reaction and direct sequencing of amplified E-cadherin complementary DNA fragments revealed inframe skipping of either exon 8 or exon 9 in 10 patients with diffuse tumors and an exon 9 deletion in one patient with a mixed carcinoma; both exons encode putative calcium binding domains. These alterations were not seen in nontumorous gastric tissues. Splice site mutations responsible for the exon deletions were identified in six of these patients, eliminating the possibility of alternative splicing mechanisms. Five of these splice site alterations were confirmed as somatic mutations. Non-splice site mutations were observed in three diffuse type tumors, namely a 69-base pair deletion of exon 10 and two point mutations, one of which destroys a putative calcium binding region. Immunohistochemical evaluation showed E-cadherin immunoreactivity in tumors and lymph node metastases of patients expressing abnormal mRNA. The allelic status of the E-cadherin gene was analyzed in one patient, revealing loss of heterozygosity with retention of a mutated E-cadherin allele. Overall, E-cadherin mutations were identified in 50% (13 of 26) of the diffuse type and in 14% (1 of 7) of the mixed carcinomas. In contrast, two silent E-cadherin mutations (not changing the amino acid sequence) were detected in two tumors of the intestinal type. Our study provides strong in vivo evidence that E-cadherin gene mutations may contribute to the development of diffusely growing gastric carcinomas and support a tumor/metastasis suppressor gene hypothesis.

Thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.

Parathyroid hormone stimulation of mitosis in rat thymic lymphocytes is independent of cyclic AMP.

The in vitro mitotic response of rat thymic lymphocytes to hPTH(1-34), hPTH (1-38), and 8,18 Nle hPTH(1-34) exhibits a dependency upon extracellular calcium. Removal of extracellular calcium or the addition of Verapamil (5 micrograms/ml) or trifluoroperazine (10 microM) abrogated the mitotic response. Mitogenic concentrations of 8,18 Nle hPTH(1-34) increased calcium 45 uptake from 4.49 +/- 0.25 to 8.23 +/- 0.75 pMol/10(6) cells/min. The intracellular calcium concentration, measured by Quin 2 fluorescence, also increased after addition of 8,18 Nle hPTH(1-34). Parathyroid hormone-induced activation could not be demonstrated in an otherwise responsive thymocyte membrane adenylate cyclase. In intact cells mitogenic levels of 8,18 Nle hPTH(1-34) decreased intracellular cyclic AMP content. This response was blocked by both 3-isobutyl 1-methyl xanthine and trifluoroperazine, and may indicate activation of calcium-dependent phosphodiesterase. We conclude that PTH stimulates thymic lymphocyte proliferation independently of cyclic AMP, and that changes in cellular calcium homeostasis are intimately involved in the action of PTH. In all of the assays employed, the hitherto antagonistic analogue 8,18 Nle 34 Tyr bPTH(3-34)amide proved to be an agonist. We postulate that the receptor utilized for this PTH action may not exhibit classical PTH structure-activity specificities.

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions.

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.

The iron-binding protein ferritin is expressed in cells of the osteoblastic lineage in vitro and in vivo.

Ferritin, a metal-binding protein responsible for maintaining the bioavailability of iron, has been demonstrated in cells of the osteoblastic lineage. Messenger RNAs encoding the light and heavy chain subunits of ferritin were detected in ROS 17/2.8, ROS 25/1, and UMR106 rat osteosarcoma cell lines, in fetal rat calvaria, and in primary cultures of rat calvarial osteoblast-like cells. In vivo, the expression of ferritin light-chain mRNA was observed in both active osteoblasts and in osteocytes. A 450-kD iron-binding protein was immunoprecipitated from ROS 17/2.8 cells by an antiferritin antiserum. This protein comigrated with native ferritin, and could be dissociated into subunits comigrating with ferritin light and heavy chains. Addition of extracellular Fe59-transferrin to cultures of ROS 17/2.8 cells resulted in the sequestration of the iron in intracellular ferritin. These observations demonstrate that cells of the osteoblastic lineage possess a functional ferritin-based iron uptake and storage system capable of regulating metal homeostasis in bone.

Mapping of a mouse mammary tumor virus integration site by retroviral LTR--arbitrary polymerase chain reaction.

The de novo integration of retroviral genomes within the mammalian genome is believed to contribute to the tumorigenic process. Integration may result in the disruption or inappropriate transcription of key regulatory genes. We describe the application of an arbitrarily primed PCR method for the mapping and cloning of genomic integration sites of the mouse mammary tumor virus (MMTV). We have amplified DNA sequences between a selected retroviral MMTV-LTR and random sites in the 3' flanking DNA. Using this technique we were able to visualize several proviral integration sites present in a MMTV-induced mammary tumor derived cell line that were absent from the germ line. Cloning and sequencing of the PCR product corresponding to one site established its identification as an unique 3' flanking sequence.

Some effects of parathyroidectomy on cell-mediated immune responses in the rat.

Xenografts of mouse tail skin to the rib cages of normal and sham-parathyroidectomized rats caused an increase in plasma calcium concentration and concomitant increase in bone marrow mitosis. Neither was elicited in aparathyroid rats and graft survival was prolonged in these animals. No hypercalcaemic episode was associated with the delayed hypersensitivity response induced by painting rat ears with oxazolone. Compared with the response in sham-parathyroidectomized rats, that in parathyroidectomized rats was enhanced although both responses were less than that in normal rats. Parathyroidectomy of parental donors did not affect the ability of their splenic lymphocytes to mount a graft-versus-host response in F1 hybrid recipients. When sham-operated and aparathyroid parents were sensitized with F1 hybrid lymphocytes no differences were observed in a subsequent graft-versus-host response in F1 recipients. However, when aparathyroid F1 recipients were employed a marked reduction in the graft-versus-host reaction was observed. Thus the clonal expansion of cells with specific reactivity to certain antigens in secondary lymphoid tissue, which is driven by those same specific antigens, is not affected or only moderately affected by the parathyroid status of the animal. However, the more general increase in lymphocyte numbers promoted by non-specific mitogenic lymphokines is markedly impaired in the hypocalcaemic parathyroidectomized rat. Furthermore, the parathyroid gland is essential for the development of a hypercalcaemic episode which follows antigenic challenge and causes cell proliferation in primary lymphoid tissues. This surge of mitosis could serve to replenish the depleted pools of virgin T and B lymphocytes in the secondary lymphoid tissue which occur as a result of their response to antigens.

Changes in plasma levels of calcium and in bone marrow mitosis after antigenic challenge in rats and mice.

When rats or mice were immunized with sheep red blood cells, bacterial lipopolysaccharides or bovine serum albumin, a proliferative response could be detected in the bone marrow and spleen. This response was associated with a hypercalcaemic phase. Parathyroidectomy, which resulted in a protracted hypocalcaemia, prevented the development of an increase in levels of plasma calcium. This operation also prevented the rise in bone marrow proliferations following antigenic challenge, but did not ablate the normal proliferative response to antigen by cells in the spleen. Antibody production and numbers of antibody-forming cells were not significantly reduced by parathyroidectomy. These results suggest that there is a pool of antigen-insensitive cells in the bone marrow which are stimulated after antigenic challenge. It is postulated that these events were mediated by the development of a parathyroid-dependent hypercalcaemia which stimulates the cells non-specifically. These events may form part of a cellular homeostasis, replacing cells in peripheral lymphoid tissues.

Circannual variations in serum concentrations of pituitary, thyroid, parathyroid, gonadal and adrenal hormones in male laboratory rats.

Seasonal effects were studied on basal levels of hormones in the serum of adult male Sprague-Dawley rats, which were born and raised under rigorously controlled laboratory conditions. Groups of 90-day-old rats were killed at monthly intervals by rapid decapitation. Significant fluctuations were observed throughout the observation period of 19 months in serum levels of TSH, prolactin, androgens, tri-iodothyronine and LH. Minor fluctuations were observed in serum levels of FSH, corticosterone, parathyroid hormone and thyroxine. The results indicate that male laboratory rats exhibit circannual and semi-annual fluctuations in serum levels of several hormones even though the animals were born, raised and maintained in constant laboratory conditions.

The intracellular domain of cadherin-11 is not required for the induction of cell aggregation, adhesion or gap-junction formation.

The cadherin family of cell adhesion molecules demonstrates calcium-dependent homophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type I cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind beta-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.