Breast cancer tissue slices as a model for evaluation of response to rapamycin.

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical "ex-vivo" personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 µm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.

Transcriptional effects of 1,25 dihydroxyvitamin D(3) physiological and supra-physiological concentrations in breast cancer organotypic culture.

BACKGROUND: Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)(2)D(3) (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)(2)D(3) in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)(2)D(3) at concentrations that can be attained in vivo. METHODS: Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)(2)D(3) 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)(2)D(3) 0.5nM, using RT-qPCR, western blot or immunocytochemistry. RESULTS: 1,25(OH)(2)D(3) 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)(2)D(3) near physiological concentration. Genes up-modulated by both 1,25(OH)(2)D(3) concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)(2)D(3) was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)(2)D(3) 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)(2)D(3) 0.5nM was detected. CONCLUSIONS: In breast cancer specimens a short period of 1,25(OH)(2)D(3) exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)(2)D(3) effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma.

BACKGROUND: Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. MATERIAL AND METHODS: We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. RESULTS: Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN +) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN + with an accuracy of 80.0% (p = 0.012). CONCLUSIONS: The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients.

BACKGROUND: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). PATIENTS AND METHODS: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFbeta1, TGFbeta2, TGFbeta3 in oral SCC. RESULTS: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFbeta isoforms, we found that Smad2 mRNA and TGFbeta1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFbeta1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. CONCLUSION: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFbeta signaling should be better clarified in the future.

Gene expression profiles in breast cancer to identify estrogen receptor target genes.

The estrogens play important role in the homeostatic maintenance of several target tissues including those in the mammary gland, uterus, bone, cardiovascular system, and brain. Most of estrogen's action is thought to be mediated through its nuclear estrogen receptors, ERalpha and ERbeta, which are members of the nuclear receptor superfamily that act as ligand-induced transcription factors. Acting via its receptors, estrogen also plays an essential role in the development and progression of human breast cancer. The ER and progesterone receptor (PR), which are regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. However, the prognosis of a patient with ER+/PR+ breast cancer can be highly variable and a significant proportion of hormone receptor positive breast cancers does not respond to endocrine therapy. The identification of estrogen receptor target genes may improve our understanding of the role played by estrogens in breast cancer making it possible to better tailor hormone treatments and improve a patient's response to hormonal therapy. In this review, we explore the literature for data regarding the identification of estrogen receptor-regulated genes in breast cancer cell lines and breast tumor biopsies using high throughput technologies such as serial analysis of gene expression (SAGE) and cDNA microarrays.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow.

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine.

OBJECTIVES: To examine the effects of triiodothyronine (T3), 17beta-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. METHODS AND RESULTS: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3- mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. CONCLUSION: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2.

Gene stage-specific expression in the microenvironment of pediatric myelodysplastic syndromes.

Using cDNA microarray assays we have observed a clear difference in the gene expression pattern between bone marrow stromal cells obtained from healthy children (CT) and from pediatric patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) associated with MDS (MDS-AML). The global gene function profiling analysis indicated that in the pediatric MDS microenvironment the disease stages may be characterized mainly by underexpression of genes associated with biological processes such as transport. Furthermore, a subset of downregulated genes related to endocytosis and protein secretion was able to discriminate MDS from MDS-AML.

C-erbB-2 expression is a better predictor for survival than galectin-3 or p53 in early-stage breast cancer.

The definition of high risk patients with early stage breast cancer is still controversial. We evaluated the ability of galectin-3, c-erbB-2 and p53 immunohistochemical expression to predict recurrence and survival in a homogeneous set of 92 patients with T1N0M0 ductal carcinoma with a long-term follow-up. In normal breast tissue, the epithelial and fibroblast components were positive for galectin-3 mostly showing nuclear and cytoplasmic reactivity. At the tumor epithelial component, galectin-3 expression was found in 46.7% of the samples with a predominant cytoplasmic staining. Similar results were presented by concurrent in situ lesions. Tumor stromal fibroblasts maintained positivity in 70 out of 92 cases (76%). We found expression of p53 in only 16 cases (17.4%), and c-erbB-2 in 17 (18.48%). A marginal association was found between co-expression of p53 and galectin-3 (p=0.055) and a significant correlation between p53 accumulation and c-erbB-2 expression (p=0.009). There was no significant association between galectin-3 protein expression with disease-free survival or overall survival. C-erbB2 and p53 expression correlated with recurrence (p=0.002, p=0.02; respectively). Diminished overall survival at 10 years was associated with c-erbB-2 (p=0.010), but marginally with p53 expression (p=0.076). Epithelial galectin-3 expression cannot be considered a prognostic factor for patients with T1N0M0 breast cancer, p53 seems to be of minor relevance and c-erbB-2 expression was the best discriminator and may be a marker for aggressive clinical behavior in patients with early stage breast cancer.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Independent prognostic value of laminin receptor expression in breast cancer survival.

We studied the possible prognostic role of laminin, estrogen (ER), and progesterone (PR) receptors and other pathological factors in relation to the disease-free interval and overall survival of female breast carcinoma patients. Multivariate analyses of clinical and pathological data with respect to the above survival time variables were performed by Cox regression. The statistical dependence of prognosis on ER, PR, and tumor size was based on the discriminant cutoff value that could best distinguish between survival curves. Axillary nodal status was the most significant independent factor in the prediction of both disease-free interval and overall survival of these patients. Use of the information on laminin receptor expression, PR concentration, tumor size, lymphocytic infiltrate, and tumor necrosis improved significantly the prediction of the risk of recurrence. Patients with tumors expressing laminin receptors had 40% less risk of recurrence (P = 0.0209) than those with no expression. On the other hand, four covariates were independently predictive of the risk of death: axillary nodal status, lymphocytic infiltrate, PR and ER concentration. There was a marginally significant (10% level) interaction between tumor size and lymphocytic infiltrate with respect to the prediction of the risk of recurrence. The above sets of variables were used to classify patients into risk groups for the prediction of recurrence and death.

A Set of miRNAs, Their Gene and Protein Targets and Stromal Genes Distinguish Early from Late Onset ER Positive Breast Cancer.

UNLABELLED: Breast cancer (BC) in young adult patients (YA) has a more aggressive biological behavior and is associated with a worse prognosis than BC arising in middle aged patients (MA). We proposed that differentially expressed miRNAs could regulate genes and proteins underlying aggressive phenotypes of breast tumors in YA patients when compared to those arising in MA patients. OBJECTIVE: Using integrated expression analyses of miRs, their mRNA and protein targets and stromal gene expression, we aimed to identify differentially expressed profiles between tumors from YA-BC and MA-BC. METHODOLOGY AND RESULTS: Samples of ER+ invasive ductal breast carcinomas, divided into two groups: YA-BC (35 years or less) or MA-BC (50-65 years) were evaluated. Screening for BRCA1/2 status according to the BOADICEA program indicated low risk of patients being carriers of these mutations. Aggressive characteristics were more evident in YA-BC versus MA-BC. Performing qPCR, we identified eight miRs differentially expressed (miR-9, 18b, 33b, 106a, 106b, 210, 518a-3p and miR-372) between YA-BC and MA-BC tumors with high confidence statement, which were associated with aggressive clinicopathological characteristics. The expression profiles by microarray identified 602 predicted target genes associated to proliferation, cell cycle and development biological functions. Performing RPPA, 24 target proteins differed between both groups and 21 were interconnected within a network protein-protein interactions associated with proliferation, development and metabolism pathways over represented in YA-BC. Combination of eight mRNA targets or the combination of eight target proteins defined indicators able to classify individual samples into YA-BC or MA-BC groups. Fibroblast-enriched stroma expression profile analysis resulted in 308 stromal genes differentially expressed between YA-BC and MA-BC. CONCLUSION: We defined a set of differentially expressed miRNAs, their mRNAs and protein targets and stromal genes that distinguish early onset from late onset ER positive breast cancers which may be involved with tumor aggressiveness of YA-BC.

The effects of urban particulate matter on the nasal epithelium by gender: An experimental study in mice.

Nose is the first portion of the respiratory system into contact with air pollution particles, including organic compounds that could act as endocrine releasers. The objective was to identify and quantify estrogenic receptor-ß (ERß), aryl hydrocarbon receptor (AhR), the cytochrome P450 enzymes CYP1A1, 1A2, 1B1, and mucus profile in the nasal epithelium of mice. BALB/c mice male (n = 32) and female (n = 82) in proestrus, estrus and diestrus were divided into two groups: 1) exposed to ambient air; 2) concentrated ambient particles (CAPs) to achieve an accumulated dose (concentration vs. time product) of 600 µg/m(3), the time of the exposure was controlled to ensure the same concentration for all groups (5 days per week for 40-51 days). RT-PCR (Erß-1, Erß-2, Ahr, Cyp1a1, Cyp1a2, Cyp1b1), immunohistochemistry and morphometry (ERß, AhR) were used to analyze. The mucus profiles were examined using acid (Alcian Blue) and neutral (periodic acid Schiff's) stains. Exposed females had significantly lower levels of Erß-2 mRNA than exposed males (p = 0.036). Cyp1b1 mRNA in diestrus females was significantly lower in the CAP-exposed group compared with the ambient air group (p ≤ 0.05). ERß expression in the epithelium and submucosa nucleus was lower in estrus exposed to CAPs compared with ambient air. CAPs increases AhR in the epithelium (p = 0.044) and submucosa (p = 0.001) nucleus of female when compared with male mice. Exposure to CAPs, also led to relatively increased acidic content in the mucus of males (p = 0.048), but decreased acidic content in that of females (p = 0.04). This study revealed sex-dependent responses to air pollution in the nasal epithelium that may partially explain the predisposition of females to airway respiratory diseases.

Expression of a novel factor, com1, in early tumor progression of breast cancer.

Tumor cells and their surrounding microenvironment produce a variety of growth factors and proteolytic enzymes to promote tumor growth and metastasis. We have recently identified a novel factor, termed com1, which is up-regulated in human breast carcinoma cells upon formation of experimental metastatic tumors and assumed to act as a growth-promoting factor in breast cancer. In attempts to explore the biological role of com1 in clinical tumor growth and metastasis, expression of com1 mRNA in primary carcinomas from 81 breast cancer patients and 27 samples of uninvolved adjacent breast tissue from these patients was compared and related to known prognostic parameters and outcome. The levels of com1 mRNA were significantly up-regulated (P < 0.0001) in the tumors compared to the normal breast tissues. Tumor expression of com1 mRNA, however, did not correlate with the mRNA levels for urokinase-type plasminogen activator (uPA), its receptor, or the type 1 inhibitor, which are factors that define a phenotype of tumor aggressiveness when elevated. And whereas the mRNA levels of uPA and the uPA receptor were elevated in tumors from the patients who subsequently had poor outcome, no correlations were observed between tumor com1 mRNA expression and prognosis or histological and biochemical characteristics of the tumors. We therefore assume that com1 may mediate some growth-promoting function early in development of the primary breast carcinoma, but not in later stages of tumorigenesis or metastasis.

A role for the laminin receptor in leukocyte chemotaxis.

Previous studies on the mechanism of leukocyte traversal of basement membranes showed that rabbit peritoneal exudate polymorphonuclear cells (PMN) preferentially used laminin, a major constituent of basement membrane, to attach to another component, type IV collagen. PMN also responded chemotactically to nanomolar levels of laminin. We have now determined that PMN possess a receptor for laminin. Scatchard analysis using 125I laminin indicates a single class of saturable high affinity binding sites (kd = 6.15 nM/L) on PMN and 3.6 X 10(4) sites per cell. A chymotryptically derived fragment of laminin, C1, which lacks both the long arm and the globular end regions of the short arm (i.e., matrix binding sites) but retains the laminin receptor binding region, gave similar results. Immunoperoxidase studies using monoclonal antibodies to the laminin receptor (mAbLR) indicated the presence of the receptor on the surface of PMN. These cells responded chemotactically to nanomolar levels of C1 and laminin, a result consonant with binding data. PMN chemotaxis to a formyl peptide was markedly inhibited by mAbLR, suggesting that the laminin receptor may be required for PMN chemotaxis in general. Our results suggest that PMN extravasation across basement membranes is aided both by reversible attachment of the cells to laminin in the matrix and by chemotaxis to a gradient of soluble intact and possibly degraded laminin. These characteristics have much in common with those of highly metastatic tumor cells.

Profile of thyroid hormones in breast cancer patients.

Estrogen involvement in breast cancer has been established; however, the association between breast cancer and thyroid diseases is controversial. Estrogen-like effects of thyroid hormone on breast cancer cell growth in culture have been reported. The objective of the present study was to determine the profile of thyroid hormones in breast cancer patients. Serum aliquots from 26 patients with breast cancer ranging in age from 30 to 85 years and age-matched normal controls (N = 22) were analyzed for free triiodothyronine (T3F), free thyroxine (T4F), thyroid-stimulating hormone (TSH), antiperoxidase antibody (TPO), and estradiol (E2). Estrogen receptor ss (ERss) was determined in tumor tissues by immunohistochemistry. Thyroid disease incidence was higher in patients than in controls (58 vs 18%, P < 0.05). Subclinical hyperthyroidism was the most frequent disorder in patients (31%); hypothyroidism (8%) and positive anti-TPO antibodies (19%) were also found. Subclinical hypothyroidism was the only dysfunction (18%) found in controls. Hyperthyroidism was associated with postmenopausal patients, as shown by significantly higher mean T3 and T4 values and lower TSH levels in this group of breast cancer patients than in controls. The majority of positive ERss tumors were clustered in the postmenopausal patients and all cases presenting subclinical hyperthyroidism in this subgroup concomitantly exhibited Erss-positive tumors. Subclinical hyperthyroidism was present in only one of 6 premenopausal patients. We show here that postmenopausal breast cancer patients have a significantly increased thyroid hormone/E2 ratio (P < 0.05), suggesting a possible tumor growth-promoting effect caused by this misbalance.

Expression of gelatinases A and B, stromelysin-3 and matrilysin genes in breast carcinomas: clinico-pathological correlations.

The purpose of this study was to investigate the association among matrix metalloproteinases (gelatinases A and B, stromelysin-3 (ST3) and matrilysin) mRNAs expressed in primary breast carcinomas and standard prognostic parameters and clinical outcome. mRNA levels were determined by Northern analysis in samples of 81 breast cancer patients (median follow-up, 40 months) and 27 samples of uninvolved adjacent breast tissue. Proteases were expressed by the majority of the tumors and normal breast tissues examined. ST3, gelatinase A and matrilysin mRNAs were more often expressed at high levels in carcinomatous than in normal breast tissues. Differences in the distribution of gelatinase B mRNA were not found. However, paired normal tissues generally produced weaker signals when compared to matched tumor samples. Univariate analysis showed no significant association of gelatinase A and matrilysin mRNAs with the classical prognostic markers (age, menopausal status, stage, size, nodal status, vascular infiltrate, necrosis, steroid receptors, metastasis and survival). Overexpression of ST3 was more frequently found in tumors of post-menopausal women (P < 0.022). Elevated expression of gel B mRNA was associated with the presence of vascular infiltrate (P < 0.026), necrosis (P < 0.039), PR negative tumors (P < 0.014) and inversely correlated to the number of survivors (P < 0.021). Multivariate analysis including 68 patients for whom all information was available indicated that neither stromelysin correlated significantly with pathological, clinical or biochemical features. High levels of gelatinase A and B mRNAs were inversely associated with the number of survivors. Our findings suggest that measurements of gelatinase A and B mRNAs expression in breast carcinoma may help to identify patients with an aggressive form of the disease.

Repression of glucocorticoid receptor gene transcription by c-Jun.

The regulation of glucocorticoid receptor gene expression by members of the AP-1 family was examined in glucocorticoid-free NIH3T3 cells transfected with the human glucocorticoid receptor gene promoter driving expression of a CAT reporter gene. c-Jun inhibited the promoter activity by 80% and JunB by 30%, whereas c-Fos and JunD had no inhibitory effect. Electrophoretic mobility shift assays showed that c-Jun is unable to efficiently interact with the AP-1-like site present in the human glucocorticoid receptor promoter. Moreover, c-Jun was still able to repress promoter mutants in which the region containing the AP-1-like site was deleted. NIH3T3 cell clones overexpressing c-Jun exhibited lower glucocorticoid receptor mRNA levels, which suggests that the murine glucocorticoid receptor gene can also be regulated by AP-1. These results provide a new mechanism for cross-talk between the glucocorticoid receptor and the AP-1 family of transcription factors in the absence of glucocorticoid ligands.

Vitamin D3 binding activity during leukemic cell differentiation.

In this paper we report that differentiation of the human promyelocytic leukemia cell line, HL60, along the myelocytic pathway, induced by retinoic acid (RA), or monocytic pathway, induced by phorbol-myristate acetate (PMA) and gamma interferon (IFN), was accompanied by a significant decline in 1,25-dihydroxycholecalciferol (1,25(OH)2D3) binding (control: 30.3 +/- 3.0 fM/10(6) cells; RA treated: 6.8 + 2.5 fM/10(6) cells; PMA treated: 12.3 +/- 6.7 fM/10(6) cells and IFN treated: 16.0 +/- 5.0 fM/10(6) cells). When differentiation and proliferation were uncoupled, by incubation with IFN or by inhibition of proliferation by cell density saturation, 1,25(OH)2D3 binding was better related to differentiation than to proliferation. Additionally we have compared 1,25(OH)2D3 binding levels in blasts from acute lymphocytic leukemia (ALL) patients (25.4 +/- 18.1 fM/10(6) cells) and normal, mature lymphocytes (10.6 +/- 2.1 fM/10(6) cells). Receptor binding was significantly higher (p < 0.05) in the immature blasts. Our data suggest that 1,25(OH)2D3 receptor levels could be considered a marker of functional immaturity, in these cells.

Bone marrow stroma in childhood myelodysplastic syndrome: composition, ability to sustain hematopoiesis in vitro, and altered gene expression.

We studied bone marrow stromal cell cultures from patients with childhood myelodysplastic syndromes (MDS, refractory anemia with excess of blasts, RAEB) and from matched normal donors. Stromal cell monolayers were characterized as myofibroblasts by the expression of smooth muscle alpha-actin, collagen IV, laminin and fibronectin. When normal cord blood cells were plated onto myelodysplastic stromas, a pathologic cell differentiation was observed, indicating altered myelosupportive properties. cDNA array analysis showed that patient stromas expressed increased levels of thrombospondin-1, collagen-I alpha2-chain, osteoblast-specific factor-2 and osteonectin, indicating the presence of increased osteoblast content, as confirmed by enhanced alkaline phosphatase synthesis. Alterations in the myelodysplastic stroma environment might contribute to abnormal hematopoiesis in this pathology.