Evaluation of HER2, p53, bcl-2, topoisomerase II-alpha, heat shock proteins 27 and 70 in primary breast cancer and metastatic ipsilateral axillary lymph nodes.

BACKGROUND: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. OBJECTIVE: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. PATIENTS AND METHODS: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 microm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel-Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-alpha (clone KiS1, 1 microg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CB11, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. RESULTS: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-alpha, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2, 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-alpha and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-alpha, 22% for HSP27 and 25% for HSP70. CONCLUSIONS: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.

Interstitial insertion of varying amounts of ABL-containing genetic material into chromosome 22 in Ph-negative CML.

We studied the cells from three selected patients with Ph-chromosome-negative chronic myeloid leukemia (CML) by Southern blotting, polymerase chain reaction, and in situ hybridization of informative probes to metaphase chromosomes. All three patients had rearrangement of M-BCR sequences in the BCR gene and expression of one or other of the mRNA species characteristic of Ph-positive CML. Leukemic metaphases studied after trypsin-Giemsa banding were indistinguishable from normal. The ABL probe localized both to chromosome 9 and 22 in each case. A probe containing 3' M-BCR sequences localized only to chromosome 22, and not to chromosome 9 as would be expected in Ph-positive CML. Two new probes that recognize different polymorphic regions distal to the ABL gene on chromosome 9 in normal subjects localized exclusively to chromosome 9 in two patients and to both chromosomes 9 and 22 in one patient. These results show that Ph-negative CML with BCR rearrangement is associated with insertion of a variable quantity of chromosome 9 derived material into chromosome 22q11; there is no evidence for reciprocal translocation of material from chromosome 22 to chromosome 9.

The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth.

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

Genomic organization of the human e1af gene, a member of Ets transcription factors

The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19 kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7 kb. The 5'-untranslated region is about 0.3 kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF. (C) 1999 Elsevier Science B.V. All rights reserved.

Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

Intrastereomic proteins from the asteroid Asterias rubens. separation, partial characterization ,and interactions with growing calcite crystals

info:eu-repo/semantics/published

On the structural alterations of phage proteins synthesized in the presence of pancreatic ribonuclease.

Journal Article

Normal IgG response to dermatophagoïde P1 antigen, bovine betalactoglobulin and soya proteins

info:eu-repo/semantics/published

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guerin (BCG) in patients with superficial bladder cancer

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgC antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post- treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P < 0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013

The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100 % of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90 % of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.