Breast Cancer Subtype is Associated With Axillary Lymph Node Metastasis: A Retrospective Cohort Study.

The purpose of this study was to assess whether breast cancer subtype (BCS) as determined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 can predict the axillary lymph node metastasis in breast cancer. Patients who received breast conserving surgery or mastectomy and axillary lymph node dissection were identified from 2 cancer centers. The associations between clinicopathological variables and axillary lymph node involvement were evaluated in univariate and multivariate regression analyses. A total of 3471 patients met the inclusion criteria, and 53.0% had axillary lymph node metastases at diagnosis. Patients with hormone receptor (HR)-/human epidermal growth factor receptor 2 (HER2)- subtype had a higher grade disease and the lowest rate of lymphovascular invasion. Univariate and multivariable logistic regression analyses showed that BCS was significantly associated with lymph node involvement. Patients with the HR-/HER2- subtype had the lowest odds of having nodal positivity than those with other BCSs. HR+/HER2- (odds ratio [OR] 1.651, 95% confidence interval [CI]: 1.349-2.021, P < 0.001), HR+/HER2+ (OR 1.958, 95%CI 1.542-2.486, P < 0.001), and HR-/HER2+ (OR 1.525, 95%CI 1.181-1.970, P < 0.001) tumors had higher risk of nodal positivity than the HR-/HER2- subtype. The other independent predictors of nodal metastases included tumor size, tumor grade, and lymphovascular invasion. Breast cancer subtype can predict the presence of axillary lymph node metastasis in breast cancer. HR-/HER2- is associated with a reduced risk of axillary lymph node metastasis compared to other BCSs. Our findings may play an important role in guiding axillary treatment considerations if further confirmed in larger sample size studies.

Common protein biomarkers assessed by reverse phase protein arrays show considerable intratumoral heterogeneity in breast cancer tissues.

Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2-5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22-43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range 18-38%) or between different tumor zones (CV 24%, range 17-38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18-34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29-98%) and lymph node metastases (CV 65%, range 40-146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography.

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.

Biopsy of a 5 mm cystic lesion on the right heel of a 48-year-old woman.

An excisional biopsy of an asymptomatic cystic lesion that had been present for several years on the right heel of a 48-year-old woman revealed a subcutaneous cyst lined by ciliated columnar epithelium. On immunostaining, the epithelial cells were positive for Pan-cytokeratin (CK AE 1/3), estrogen receptor (ER) and progesterone receptor (PR), but negative for carcinoembryonic antigen (CEA), suggesting Mullerian type of epithelium. Cutaneous ciliated cyst of Mullerian type occurs almost exclusively on the lower extremity of premenopausal women. The lesion is benign and excision is curative.

Electrochemical detection of oestrogen binding protein interaction with oestrogen in Candida albicans cell lysate.

Previously we reported an electrochemical method to quantitatively detect vertebrate oestrogens using wild type Saccharomyces cerevisiae cells. That assay required the use of a double mediator system, a five-hour incubation period and had a maximum detection limit of around 11 nM 17ß-oestradiol. In the work reported here we have sought to systematically increase the utility and decrease the complexity of the whole cell assay. The steps we took to achieve this goal were in order; lysing the cells to remove transport constraints, removing the lipophilic mediator and conducting the assay with the hydrophilic mediator only and finally performing the assay in a complex medium to demonstrate its specificity. Linear sweep voltammetry was used to investigate the interaction of mediators with NADH. The assay is now cell free and functions in a complex substrate. The linear response range upper limit has been raised to 100 nM with a calculated limit of detection of 0.005 nM with a limit of determination of 0.014 nM and the assay period has been reduced to 20 min.

Isolation and characterization of three estrogen receptor transcripts in Oreochromis mossambicus (Peters).

Exposure of aquatic organisms to 17beta-estradiol (E(2)) induces a variety of estrogen-responsive genes, including vitellogenin (vtg)-the precursor protein of egg yolk in oviparous animals and to date the single most used gene product in screening for estrogenic endocrine disruption. Transcription regulation of vtg by E(2) is dependent on binding of the ligand (E(2)) to a specific nuclear receptor (estrogen receptor, ESR) which in turn binds to an estrogen responsive element (ERE) in the promoter of vtg. Since a local tilapiine, Oreochromis mossambicus (Peters), is targeted as a model for estrogenic endocrine disruption in Southern Africa, a platform of knowledge is necessary for the ontogenic and tissue specific behavior of ESR in this species before vtg levels can be interpreted in relation to such endocrine disruption. Therefore, three ESR cDNA sequences (ESR1, ESR2a and ESR2b) in O. mossambicus were isolated and QPCR protocols were developed to ascertain their quantitative transcript levels in adult brain, gonadal and hepatic tissues. ESR1 transcript levels were highest in female liver tissue compared to males and other tissues, whereas the levels for ESR2a and b were not statistically significantly different between male and female tissues. Quantitative gene levels during development demonstrated a sharp increase in ESR1 during the stage of gonad differentiation (50-60 days post-fertilization) in this species. Finally, an induction experiment in adult male liver tissue confirms the upregulation of ESR1 by E(2).

Immunohistochemical assessment of hormone receptor status of breast carcinoma: interobserver variation of the quick score.

BACKGROUND: Immunohistochemical (IHC) assessment of estrogen receptor (ER) and progesterone receptor (PR) status has become a routine practice to predict the likely outcome of Tamoxifen therapy. AIMS: To assess the interobserver variation in scoring hormone receptor status of breast carcinoma, using the Quick Score. MATERIALS AND METHODS: IHC-stained slides of breast carcinomas reported by the two authors during a 28-month period were included in the study. Both authors independently reassessed all the tumors. Both were blinded to each other's assessment. The Quick score with a 0-8 point scale was used to score the hormone receptor status. Weighted Kappa was calculated to assess the interobserver variation. RESULTS: A total of 210 breast carcinomas were included in this study. There was a substantial to almost perfect agreement between the two observers in scoring the hormone receptor status (kappa values; ER=0.856, PR=0.711). Both ER and PR showed an almost perfect agreement in assessing the intensity of staining (kappa value; ER=0.882, PR=0.840), while the scoring of proportion of cells gave lower Kappa values (kappa value; ER=0.778, PR=0.592). Interobserver agreement was less in scoring hormone receptor status of breast carcinomas after mastectomies compared with excision biopsies, wide local excisions and metastatic deposits in lymph nodes. Suboptimal fixation resulting in background staining has contributed to the variation. CONCLUSION: A substantial to almost perfect interobserver agreement was seen in assigning an overall Quick score. Detection of complete negative and strong expression had a moderate to substantial agreement.

Prognostic significance of molecular classification of breast invasive ductal carcinoma.

OBJECTIVE: Breast carcinoma classification has dramatically changed in recent years following application of molecular techniques. Immunohistochemistry can help select patients for different therapies. The objective of the present report is to determine the prognostic influence of the molecular classification of breast carcinoma with immunohistochemistry. MATERIALS AND METHODS: We have retrospectively selected a cohort of 257 patients with invasive ductal carcinoma NOS diagnosed and treated in the same hospital between 1997 and 2000. We have classified the cases in four tumor types according to the immunohistochemical expression of several markers, as luminal A tumors [estrogen and/or progesterone receptors (ER/PE) positive and Her-2 negative]; luminal B tumors (ER/PR positive and Her-2 positive); Her-2 positive tumors (ER/PR negative with Her-2 positive); and triple negative phenotype (all markers negative). RESULTS: In our series, 116 patients had tumors of luminal A type (47.93%); 67 (27.68%) were luminal type B; 33 (13.63%) were Her2 positive; and 26 (10.74%) were triple negative. The recurrence rate was 19% for luminal type A tumors, 25.4% for luminal type B, 39.4% for Her2 positive and 30.8% for triple negative lesions. The mean relapse free survival was 79.07, 73.07, 64.3 and 83,5 months for luminal A and B, Her-2 and triple negative lesions, respectively. Mortality rate reached 11.2% for luminal A tumors compared with 19.4, 33.3 and 26.9% for luminal B, Her2 and triple negative tumors, respectively. The mean overall survival for these groups was 88.42, 81.41, 77.62 and 93.6 months. CONCLUSION: Molecular classification with immunohistochemistry behaves as a significant prognosticator for breast invasive ductal carcinoma in our series of patients. The worse prognosis observed for Her2 expressing lesions may have changed after trastuzumab use.

Estrogen receptor expression and efficacy of docetaxel-containing adjuvant chemotherapy in patients with node-positive breast cancer: results from a pooled analysis.

PURPOSE: Several adjuvant chemotherapy trials suggested that cytotoxic treatment is less effective in patients with estrogen receptor (ER)-positive breast cancers. The aim of the present study was to assess the efficacy of adjuvant docetaxel and anthracycline therapy according to ER expression in two randomized clinical trials. PATIENTS AND METHODS: Pooled data from two randomized trials, BCIRG001 and PACS01, were examined. Hazard ratios for recurrence and survival were estimated by Cox proportional hazards models and were adjusted for clinical variables. Interaction between docetaxel and ER expression was tested. RESULTS: ER status was available for 3,329 patients (95% of all randomly assigned patients), of whom 75% (n = 2,493) were ER positive. Docetaxel therapy was associated with a 30% reduction in the risk of death (hazard ratio [HR] = 0.70; 95% CI, 0.54 to 0.91) in ER-positive patients and a 31% reduction (HR = 0.69; 95% CI, 0.52 to 0.94) in ER-negative patients. Docetaxel therapy was associated with a 21% reduction in the risk of recurrence (HR = 0.79; 95% CI, 0.66 to 0.93) in ER-positive patients and a 31% reduction (HR = 0.69; 95% CI, 0.54 to 0.97) in ER-negative patients. The interaction between docetaxel therapy and ER status was not statistically significant for either overall survival (P = .87) or disease-free survival (P = .30). ER expression was also not predictive for docetaxel efficacy when it was analyzed as a semi-continuous variable based on percent of positive cells by immunohistochemistry (test for heterogeneity, P = .56 and .86 for overall survival and disease-free survival, respectively). CONCLUSION: In the pooled analysis of these two trials, docetaxel did not have a statistically significantly different effect on the risk of recurrence or death in ER-positive and ER-negative patients.

GNL3L inhibits activity of estrogen-related receptor gamma by competing for coactivator binding.

Guanine nucleotide binding protein-like 3 (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1-null yeasts. To determine the unique function of GNL3L, we identified estrogen-related receptor gamma (ERRgamma) as a GNL3L-specific binding protein. GNL3L and ERRgamma are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRgamma requires the intermediate domain of GNL3L and the AF2-domain of ERRgamma. Gain-of- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRgamma. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRgamma by GNL3L through coactivator competition.

Oyster estrogen receptor: cDNA cloning and immunolocalization.

A cDNA encoding the Pacific oyster, Crassostrea gigas, estrogen receptor (cgER) was cloned using degenerate PCR primers. The open reading frame predicted 485 amino acid residues. Comparisons of the amino acid sequence of cgER with other mollusk ERs show high similarities of the C domain (95-97%), and the E domain (56-66%). The amino acid sequence of the C domain of cgER shows 86 and 89% identity with the respective sequences of human ER-alpha and ER-beta. The amino acid sequence of the E domain of cgER shows 45% identity with those of human ER-alpha and ER-beta. The phylogenetic analysis indicated that the cgER is an ortholog of the other mollusk ERs. In the C domain, the positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. The cgER mRNA was expressed in various tissues including the ovary. Reporter gene assay revealed that cgER is unresponsive to estrogen. This result is similar to those of other mollusk ERs. ER immunoreactivity was localized mainly in the nuclei of follicle cells, the site of vitellogenin synthesis, and in oocytes. This result suggests that cgER could work as a nuclear receptor.

Tissue preferential expression of estrogen receptor gene in the marine snail, Thais clavigera.

Sex steroid hormones have been widely detected in molluscs, and experiments have shown the importance of sex steroids in sex determination, gonadal tissue maturation and gametogenesis. Nevertheless, the signaling pathways of sex steroids in invertebrates have not yet been elucidated. In order to gain insights into the mechanism of sex steroid signaling in molluscs, we have, therefore, tried to isolate molluscan estrogen receptors from the prosobranch mollusc Thais clavigera. Cerebral ganglia of T. clavigera (Mollusca, Gastropoda, Prosobranchia) were subjected to RNA extraction, and degenerate primers for amino acid sequences conserved in vertebrate estrogen receptors were designed. PCR amplification using cerebral RNA and degenerate primers followed by 5'- and 3'-RACE identified the cDNA encoding T. clavigera estrogen receptor 1 (tcER1). The deduced amino acid sequence showed 93% identity in the DNA-binding domain and 72% identity in the ligand binding domain when compared to Aplysia estrogen receptor. Reporter gene assay revealed that tcER1 is constitutively active and unresponsive to estrogen. Quantitative analysis of the tcER1 mRNA level demonstrated the preferential expression in the ovary. Furthermore, cerebral ganglia expressed tcER1 at a high level in the spring followed by subsequent enlargement of the ovary in later seasons. These results suggest importance of tcER1 in the seasonal development of reproductive organs in T. clavigera.

Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor.

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

Optimal fixation conditions for immunocytochemical analysis of estrogen receptor in cytologic specimens of breast carcinoma.

BACKGROUND: The techniques for immunostaining estrogen receptor (ER) in cytologic specimens have varied, as have the detection rates. The authors compared various fixation methods for their effect on ER detection in cytologic smears of breast carcinoma. METHODS: Smears were prepared by gently scraping the cut surfaces of 47 resected breast carcinoma specimens and placing immediately in 1 of the following conditions: 1) a sequence of 10% formalin-methanol-acetone fixatives at -20 degrees C (Abbott method); 2) 10% formalin at room temperature; and 3) Carnoy's fixative at room temperature and then Papanicolaou stained (Carnoy's-Pap). Destaining of Carnoy's-Pap smears (Carnoy's-dPap) was initially attempted before ER staining. One set of smears was also air-dried for 3 minutes before using the Abbott method. Smears and corresponding tissue sections were immunostained with anti-ER antibody 6F11 using a similar protocol except for antigen retrieval, which was not initially applied on cytologic slides. All the ER-negative smears that had been fixed with 10% formalin or Carnoy's-Pap were restained after antigen retrieval. Agreement between cytologic and histologic findings was expressed by both concordance and the kappa coefficient. RESULTS: ER detection in smears processed with the Abbott method correlated best with findings from tissue samples, with an overall correlation of 91.5% (kappa = 0.80). Findings from air-dried smears were less optimal (concordance, 84.4%; kappa = 0.65), followed by Carnoy's-Pap (concordance, 71.4%; kappa = 0.45), formalin (concordance, 31%; kappa = 0.05), and Carnoy's-dPap (concordance, 29.4%; kappa = 0.04). Antigen retrieval converted most of the ER-negative smears to positive (18 of 20 smears in formalin and 6 of 8 smears in Carnoy's-Pap), leading to a final concordance of 93% and kappa = 0.83 for both conditions. Antigen retrieval also led to stronger staining intensity without causing false positivity. CONCLUSIONS: Antigen retrieval was found to greatly improve ER immunodetectability and staining intensity in formalin-fixed and Carnoy's-Pap smears. The former may offer an alternative to the Abbott method because of its easiness and the latter can be reliably used in archival Pap-stained smears for retrospective analysis of ER. Air-drying, destaining Pap smear, and fixation in formalin or Carnoy's-Pap without antigen retrieval are not recommended.

Use of electric bedding devices and risk of breast cancer in African-American women.

In this case-control study, the authors aimed to examine whether use of an electric bedding device increased breast cancer risk in African-American women. Cases were 304 African-American patients diagnosed with breast cancer during 1995-1998 who were aged 20-64 years and lived in one of three Tennessee counties. Controls were 305 African-American women without breast cancer who were selected through random digit dialing and frequency-matched to cases by age and county. Information on the use of an electric blanket or heated water bed and other risk factors was collected through telephone interviews. Breast cancer risk associated with use of an electric bedding device increased with the number of years of use, the number of seasons of use, and the length of time of use during sleep. When women who used an electric bedding device for more than 6 months per year (and therefore were more likely to have used a heated water bed, which generates lower magnetic fields) were excluded, the corresponding dose-response relations were more striking. Similar trends in dose response were shown in both premenopausal and postmenopausal women and for both estrogen receptor-positive and estrogen receptor-negative tumors. The use of electric bedding devices may increase breast cancer risk in African-American women aged 20-64 years. Such an association might not vary substantially by menopausal status or estrogen receptor status.

Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling.

Receptors for sex and adrenal steroid hormones are absent from fully sequenced invertebrate genomes and have not been recovered from other invertebrates. Here we report the isolation of an estrogen receptor ortholog from the mollusk Aplysia californica and the reconstruction, synthesis, and experimental characterization of functional domains of the ancestral protein from which all extant steroid receptors (SRs) evolved. Our findings indicate that SRs are extremely ancient and widespread, having diversified from a primordial gene before the origin of bilaterally symmetric animals, and that this ancient receptor had estrogen receptor-like functionality. This gene was lost in the lineage leading to arthropods and nematodes and became independent of hormone regulation in the Aplysia lineage.

Identification of a novel inhibitor of breast cell growth that is down-regulated by estrogens and decreased in breast tumors.

Lifetime exposure to estrogens is a major risk factor in breast cancer, but the mechanism for this action is not fully defined. To better determine this mechanism, the activation domain of estrogen receptor (ER) alpha was used in yeast two-hybrid screenings. These screenings resulted in the identification of a novel antiproliferative protein, estrogen down-regulated gene 1 (EDG1), of which the mRNA and protein were shown to be down-regulated directly by estrogens. Our studies additionally suggested an important role for EDG1 in ER alpha-mediated breast cancer development. Analysis of 43 invasive breast cancer samples and 40 adjacent normal breast samples demonstrated EDG1 protein levels to be significantly higher in normal breast epithelial tissue as compared with breast epithelial tumor tissue. EDG1 expression levels were also correlated with the proliferation activity and ER alpha status of the tumors to examine the prognostic value of EDG1 in invasive breast tumors. EDG1 expression was more disassociated from proliferative activity as compared with ER alpha expression in tumor cells. A growth regulatory function for EDG1 is additionally indicated by studies wherein overexpression of EDG1 protein in breast cells resulted in decreased cell proliferation and decreased anchorage-independent growth. Conversely, inhibiting EDG1 expression in breast cells resulted in increased breast cell growth. Thus, we have identified a novel growth inhibitor that is down-regulated by estrogens and colocalizes with ER alpha in breast tissue. These studies support a role for EDG1 in breast cancer.

Serum p53 antibodies in correlation to other biological parameters of breast cancer.

Breast cancer is the second most frequent cancer of Thai women. Mutation of p53 is a common event in breast cancer. This alteration can result in cellular accumulation of p53 and may also found in serum p53 antibodies (p53-Abs). To clarify prognostic significance of these antibodies, we evaluated p53-Abs in 158 sera of patients with breast cancer. Thirty (19%) patients were found to have p53-Abs. The incidence of p53-Abs tended to be higher in patients with advanced disease group (stages III and IV) than patients with early disease group (stages I and II) (P=0.055). Strong correlations were found between the presence of p53-Abs and p53 protein expression (P<0.001) and lymph node status (P=0.021). The presence of p53-Abs was associated with lack of estrogen (ER) receptor expression (P=0.035) but was not related to progesterone receptor (PR) (P=0.567). In addition, there was a statistically significant correlation between p53-Abs and proliferation associated antigen Ki-67 (P=0.006), but no relation between c-erbB2 oncoprotein and p53-Abs was observed (P=0.112). Additionally, no correlation was noted between the presence of p53-Abs and serum carcinoembryonic antigen (CEA) or carbohydrate antigen (CA15-3). Our findings indicate that p53-Abs appears to be a promising new parameter to evaluate the cellular biology and prognosis of breast cancer.

Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol covalent attachment sites on estrogen receptor alpha.

Mass spectrometry was used to identify the sites of covalent attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The covalent attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.