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Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

In breast cancer patients, primary chemotherapy is associated with the same survival benefits as adjuvant chemotherapy. Residual tumors represent a clinical challenge, as they may be resistant to additional cycles of the same drugs. Our aim was to identify differential transcripts expressed in residual tumors, after neoadjuvant chemotherapy, that might be related with tumor resistance. Hence, 16 patients with paired tumor samples, collected before and after treatment (4 cycles doxorubicin/cyclophosphamide, AC) had their gene expression evaluated on cDNA microarray slides containing 4,608 genes. Three hundred and eighty-nine genes were differentially expressed (paired Student's t-test, pFDR<0.01) between pre- and post-chemotherapy samples and among the regulated functions were the JNK cascade and cell death. Unsupervised hierarchical clustering identified one branch comprising exclusively, eight pre-chemotherapy samples and another branch, including the former correspondent eight post-chemotherapy samples and other 16 paired pre/post-chemotherapy samples. No differences in clinical and tumor parameters could explain this clustering. Another group of 11 patients with paired samples had expression of selected genes determined by real-time RT-PCR and CTGF and DUSP1 were confirmed more expressed in post- as compared to pre-chemotherapy samples. After neoadjuvant chemotherapy some residual samples may retain their molecular signature while others present significant changes in their gene expression, probably induced by the treatment. CTGF and DUSP1 overexpression in residual samples may be a reflection of resistance to further administration of AC regimen.

Objective: To better understand the estrogen (E2) agonist action of triiodothyronine (T3) the effects of these hormones on ER negative MDA-MB-231 breast cancer cells were compared with those on S30, a clone of MDA-MB-231 stably transfected with ERalpha cDNA, in terms of proliferation and modulation of hormone receptors., Results: Growth experiments showed that MDA-MB-231 was not modulated by any hormone or tamoxifen (TAM). Treatment with E2, 10(-8)M or 10(-9)M had little effect on S30 proliferation. T3 at 10(-8)M significantly inhibited proliferation. This effect was not reverted by TAM. Treatments with 10(-8)M concentration of E2 or T3 reduced ERalpha gene expression in S30, an effect partially blocked by association with TAM, with no effect on TR expression. These results suggest that, in S30, 10(-8)M T3 has a similar action to E2 relative to ERalpha gene modulation., Conclusions: Such results emphasize the need of determining T3 levels, before the introduction of antiestrogenic forms of treatment in breast cancer patients.

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 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.

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro., Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 muM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples > or = 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors., Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction > or = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed., Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

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.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare benign neoplasm of the nasopharynx that accounts for 0.5% of all head and neck tumors. Although histologically benign in appearance, JNAs are locally aggressive and destructive, spreading from the nasal cavity to the nasopharynx, paranasal sinuses, and orbit skull base with intracranial extension. The gender selectivity of JNA and the relatively young age at diagnosis suggest hormone-dependent development. Hormonal disorders have been reported in patients with JNA, and androgen and estrogen receptors have been identified in tumor tissue; however, a hormonal influence on JNA is controversial. Recent studies have attempted to further delineate the pathogenesis of JNA through analysis of genetic and molecular changes. Understanding of the molecular mechanisms involved in JNA might improve prevention, prognosis, and treatment of this tumor. In this review, we discuss published studies addressing the possible molecular pathways that might be involved in the development of JNA., ((c) 2008 Wiley Periodicals, Inc. Head Neck, 2008.)

In a previous study, using differential display reverse transcriptase-PCR (DDRT-PCR) we showed that down-regulation of the PHLDA1 (pleckstrin homology-like domain, family A, member 1; also named TDAG51) mRNA was down-regulated in breast tumors compared with normal breast tissue. The present study was conducted to determine the expression pattern and predictive prognostic value of PHLDA1 in breast cancer. A series of 720 primary invasive breast tumors were examined for PHLDA1 expression. PHLDA1 mRNA expression was determined in 74 breast tumors using quantitative Real Time PCR analysis (qPCR). PHLDA1 protein expression was evaluated by immunohistochemistry (IHC) using Tissue Microarrays (TMA) containing 699 primary invasive breast tumors. Reduced PHLDA1 mRNA expression was identified in 72% (53/74) of the primary breast tumors analyzed. Seventy-three percent (512/699) of cases analyzed showed negative PHLDA1 protein expression. Down-regulation of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer patients. Breast cancer patients with tumors that were negative for PHLDA1 protein expression had shorter disease free survival (P < 0.001) and overall survival (P < 0.001) than patients with tumors that were positive for PHLDA1 protein expression. In addition patients with tumors exhibiting reduced PHLDA1 expression and paucity for ER had the worse outcome (P < 0.001). Multivariate analysis indicated that PHLDA1 protein expression is an independent prognostic factor of patient survival. To our knowledge, the expression pattern of PHLDA1 in breast cancer has not previously been investigated. Our results provide strong evidence that reduced PHLDA1 expression is important in breast cancer progression and could serve as useful prognostic marker of disease outcome.

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.

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.

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.

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.

The members of the DnaJ/Hsp40 proteins are highly conserved through evolution, expressed in several tissues and act as co-chaperone regulating protein folding, transport, translational initiation and gene expression. Recently, using cDNA microarray we identified differences in the expression of the JDP1 (DNAJC12) gene, a member of the DnaJ/Hsp40 family, between ER-positive and ER-negative breast tumours. In this study, using quantitative real-time PCR (qPCR) we evaluated the expression pattern of the JDP1 gene in a series of 72 primary breast tumours and investigated the effects of 17beta-estradiol on the expression of the JDP1 in MCF-7 breast cancer cells. Three patterns of JDP1 mRNA expression were identified in the primary breast tumours analysed: normal expression was found in 14% of the cases, under-expression in 50%, and over-expression in 36% of the cases. High levels of JDP1 mRNA expression were significantly associated with estrogen receptor-positive status (p=0.02). No relationship was found between JDP1 mRNA expression and any other clinicopathological characteristics of the patients. Sequence analysis of the promoter region of the JDP1 gene revealed the presence of potential estrogen response elements (EREs), suggesting it to be under the control of estrogen action. We also assessed the effects of 17beta-estradiol (10 nM) on JDP1 mRNA expression in MCF-7 breast cancer cells. The JDP1 transcripts were found to be up-regulated in a time-dependent fashion in MCF-7 cells exposed to 17beta-estradiol treatment. Here we show for the first time that JDP1 is a estrogen target gene and that its expression might be used as a marker of the ER transactivation activity and may have a predictive value for response to hormonal therapy.

Purpose: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients., Experimental Design: Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays., Results: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (> or =30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one., Conclusion: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.

Prostate cancer (PC) is one of the main causes of disease and death and represents the second cause of death among men in Brazil. Benign prostate hyperplasia is a progressive and prevalent disease. It is estimated that men present around 50% and 90% of histological evidences of prostate hyperplasia at 50 and 80 years, respectively. While the pathogenesis of prostate neoplasias has been closely related to androgen and their specific nuclear receptor, the molecular mechanisms of cell growth, differentiation and apoptosis processes are still not clearly established. Co-activators and co-repressors could also contribute to prostate carcinogenesis by their binding to nuclear receptors or by interacting with the transcriptional machinery in order to increase the transcription of target genes. AR and type 2 5alpha reductase polymorphisms seem to be associated to the risk for PC. In addition, apoptosis and cellular cycle regulator genes, as well as growth factors, have been reported to be associated with the prostate tumorigenesis. Therefore, changes on the gene expression of normal tissue may be associated to the development of malign phenotype and these genes could be regarded as candidates of prognosis markers. The number of these genes increases every day but the present data needs further studies and correlation with the disease progression.

The activating protein-1 (AP-1) family of transcription factors has been implicated in the control of proliferation and differentiation of keratinocytes, but its role in malignant transformation is not clear. The aim of this study is to assess the pattern of mRNA expression of jun-fos AP-1 family members in 45 samples of head and neck squamous cell carcinomas (HNSCC) and matched adjacent mucosa by means of Northern blot analysis. Transcripts of all family members were identified, except for JunB that was detected only by means of reverse transcription polymerase chain reaction. Neither c-Fos nor JunD or FosB mRNA differed between tumours and normal tissues. We observed a strong Fos-related antigen-1 (Fra-1) and Fra-2 expression, but only Fra-1 mRNA densitometric values were higher in tumour, compared to normal adjacent mucosa (t-test, P = 0.006). A direct relationship between the positive expression of Fra-1 mRNA, above tumour median, was associated with the presence of compromised lymph nodes (Fischer exact test, P = 0.006). In addition, Fra-1 protein staining was assessed in a collection of 180 tumours and 29 histologically normal samples adjacent to tumours in a tissue array. Weak reactivity, restricted to the basal cell layer, was detected in 79% of tumour adjacent normal tissues, opposed to the intense reactivity of cancer tissues. In the subgroup of oral cancers, we have observed a shift in Fra-1 immunoreactivity, as long as the number of patients in each category, cytoplasmic or nuclear/cytoplasmic staining, was analysed (Fischer exact test, P = 0.0005). Thus, Fra-1 gene induction and accumulation of Fra-1 protein may contribute to the neoplastic phenotype in HNSCC.

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.

Estrogen acts via its receptor (ER) to stimulate cell growth and differentiation in the mammary gland. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in clinical management of breast cancer patients. Patients with ER- breast tumors have a poorer prognosis than patients with ER+ tumors. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR hybridized with cDNA microarrays containing 4,500 tumor-derived expressed sequence tags generated using the ORESTES technique. Samples of human primary breast carcinomas from 38 patients were analyzed. The experiments were performed in triplicates and data from each element were acquired by phosphoimage scanning. Data acquisition was performed using the ArrayVision software. After normalization statistical analysis was applied. In a preliminary analysis, 98 differentially expressed transcripts were identified, 46 were found to be more expressed in ER+/PR+ and 52 were found to be more expressed in ER-/PR- breast tumors. The biochemical functions of the genes in the reported expression profile are diverse and include metabolic enzymes, protein kinases, helicases, transcription factors, cell cycle regulators and apoptotic factors. ER-/PR- breast tumors displayed increased levels of transcripts of genes associated with neurodegeneration and genes associated with proliferation were found in ER+/PR+ tumors.

HC11, a spontaneously immortalized murine mammary lineage maintains features of normal cells while HC11 H-ras transformed cells (HC11 ras) are tumorigenic. Ras transformation is associated with a lower Vitamin D receptor (VDR) mRNA content. Our goal was to investigate the mechanism underlying VDR mRNA differences between these cells. Although the VDR transcriptional rate measured by run-on assays did not differ between the cells, our data suggested a pos transcriptional mechanism involving higher VDR mRNA degradation in HC11 ras cells which was not due to mutations in its 3'-UTR region since sequences of mRNA obtained from HC11 and HC11 ras cells were identical. Treatment of HC11 ras cells with a farnesyltransferase inhibitor, which prevents ras activation, causing an enhancement of VDR mRNA levels, indicating an association between the ras signaling pathway and VDR mRNA instability. The present work suggests that the decreased mRNA levels in HC11 ras cells might in part be due to an early loss of stability.

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.

Vitamin D seems to be an important determinant of prostate cancer risk and inherited polymorphisms in the 3'untranslated region of the vitamin D receptor (VDR) gene have been associated with the risk and progression of prostate cancer in some populations. We therefore studied VDR gene polymorphisms, as detected by Apal and Taql restriction fragments, in multiethnic Brazilian men (165 patients and 200 controls) for association with prostate cancer risk and parameters of disease severity (serum PSA, Gleason score and tumor stage). No statistical correlations were found. The unique ethnical background of Brazilian subjects, characterized by an extensive racial mixture of European, African-American and Native American, might have blunted any ethnic-specific significance of VDR polymorphisms. Further investigations of the associations between VDR and other genetic or environmental factors are warranted.

Urokinase plasminogen activator (uPA), its cell-bound receptor (uPAR) and its main inhibitor plasminogen activator type 1 (PAI-1) are present primarily in stromal cells in invasive breast carcinoma. The purpose of this study was to investigate the regulation by 1,25 dihydroxyvitamin-D3 (VD3) of these invasion-associated markers expressed in breast cancer tumors under organ culture, which preserves the interacting network of tumor and stromal cells. Breast carcinoma slices (30 cases), obtained using the Krumdieck tissue slicer, cultured for 48 h in the presence or absence of 100 nM vitamin D3, were embedded in formalin-fixed paraffin. uPA, uPAR, PAI-1 and VD3 receptor (VDR) were analyzed by immunohistochemistry, and their expression, detected in tumor cells and fibroblasts of the specimens, was not statistically changed by culture conditions. The proportion of cases expressing uPA, uPAR and PAI-1 was not affected by VD3 in epithelial cells, but the fraction of cases displaying strong PAI-1 reactivity in fibroblasts was reduced ( P=0.016) compared with control slices. Fibroblasts isolated from invasive ductal carcinomas and from normal breast tissues expressed higher VDR mRNA levels than epithelial cells. In cultured tumor fibroblasts, PAI-1 immunostaining and mRNA levels were reduced by VD3-limiting fibroblast contribution to invasion.

c-myc and p53 genes were frequently deregulated in head and neck squamous cell carcinoma (HNSCC). To determine if the concomitant expression of the two oncogenes might have prognostic value, the survival and free disease time of 140 consecutive HNSCC patients followed up for a median time of 29.9 months was analyzed in the light of p53 and c-myc expression assessed by immunohistochemistry. Positive c-myc and p53 staining was detected respectively in 35.7 and 50.7% of the tumors. Double positivity emerged in 16.4% of the cases. Overall-survival of patients was not associated with the immunoreactivity of p53 or c-myc considered separately or grouped in subsets. Considering only the advanced stages, the concomitant expression of both oncogenes in tumors was associated with worse disease-free survival (P = 0.004) suggesting a role for p53 and c-myc genes in progression of this HNSCC subset. Clinical parameters (presence of lymph nodes, histologic grade and tumor width) remained important indicators of overall survival (OS).

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 M 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of -casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR , as well as RXR a, and g expression was low in both HC11 and HC11ras cells. In addition, RAR mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

There is a large and increasing body of experimental and clinical data supporting the involvement of estrogen on the proliferation of hormone-dependent breast tumors. Estrogen acts via its receptor (ER) stimulating cellular proliferation. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR. Using the technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we have isolated and cloned 127 cDNA fragments that showed differential expression in either ER+/PR+ or ER-/PR- breast tumors. Sequencing analysis of these clones revealed that 119 cDNAs had homology with known sequences in the National Center of Biotechnology Information (NCBI) and 8 were novel, showing no homology to known genes. Among these differentially expressed transcripts are metabolic enzymes, ribosomal proteins, transcription factors, hypothetical proteins, cell cycle regulators, cytoskelectum related genes, cell adhesion and motility genes. Differences in gene expression profiles are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast tumors.

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of beta 1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha 2 (63.8 11.3% positive cells), alpha 3 (93.3 7.0%), alpha 5 (50.4 12.0%) and alpha 6 (34.1 4.9%) integrins but not alpha1, alpha 4, alpha v or 4. Cells adhered well to laminin-1 (73.4 6.0%) and fibronectin (40.0 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha 2, alpha 3 and alpha 6 mediated laminin-1 adhesion, but neither alpha 3 nor alpha 5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 2.4% vs DMSO: 70.7 2.5%) while simultaneously reducing alpha 5 (24.2 19%) and alpha 6 (14.3 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha 3 and alpha 5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 2 cells vs DMSO: 64 6 cells), was blocked by an antibody against alpha 6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

We have attempted to determine the incidence, nature and clinical significance of TP53 mutation in a group of white (242 cases) and African-Brazilian (52 cases) patients with breast cancer. The interethnic admixture as estimated by STR markers showed that white subjects displayed 67.9+/-0.4%, 25.0+/-1.7% and 7.0%+/-1.6% and the black populations had 34.4+/-1.9%, 56.2+/-1.9 and 9.4+/-2.2% respectively of European, African and Amerindian genes. Clinical parameters such as age, lymph node status and steroid receptors were similar in both groups. African-Brazilian patients presented more advanced lesions. Mutation screening was performed using polymerase chain reaction-single strand conformation analysis followed by sequencing. Compared to whites (13.6%), a relatively high frequency of TP53 mutation was found in blacks (32.7%) (p=0.001). African-Brazilian women have a larger proportion of mutations in exons 5 and 7, whereas white women have more mutations in exon 8. Mutations within exon 4 were found only in tumors of white patients. The spectra of TP53 mutations show that A:T-->G:C nucleotide transversion and G:C-->C:G transition were more common in African-Brazilian women whereas G:C-->T:A transversion occurs very frequently in whites. A high prevalence of G:C-->A:T nucleotide transitions and deletions was detected in both groups. No association was found between p53 gene mutation and tumor or clinical parameters independently of the ethnic group. With a median follow-up of 35.6 months for whites and 43.4 months for the blacks, no differences in overall survival were found. If white patients were stratified according to the type and location of TP53 mutations, patients with mutations affecting amino acids directly involved in DNA or Zn binding displayed a poor prognosis. The pattern of mutations found in our population seems to reflect a base line pattern observed in populations with similar ethnic profile with some modifications, which might be derived from specific etiological factors.

Background: Transforming growth factor beta1 (TGFbeta1) is a negative growth regulator in keratinocytes, and in vitro studies lead to the concept that loss of TGFbeta1 responsiveness is a critical step in epithelial carcinogenesis., Objective: To investigate the prognostic relevance of TGFbeta1 expression in head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: TGFbeta1 distribution was determined by immunohistochemistry in oral cavity/oropharynx (n = 79), larynx (n = 36) and hypopharynx (n = 25) tumors and in matched normal adjacent mucosa. TGFbeta-type I and II receptors were determined in 20 cases of differentiated oral cavity/hypopharynx tumors. Cases were considered positive if displaying reactivity in >10% of the cells., Results: TGFbeta1-positive expression was found in 47.2% of larynx, 36.7% of oral cavity/oropharynx and in 24% of the hypopharynx tumors. Reactivity in >60% of the cells was displayed only by 11.4% of HNSCC. All normal controls were positive. TGFbeta1-positive expression did not correlate with clinico pathological parameters. An association with differentiation was verified only in oral cavity/oropharynx tumors (P

IGFII and H19 genes are expressed only from one allele due to genomic imprinting, biallelic expression (loss of imprinting) being associated with the tumorigenic process of different types of tumors. The mechanism responsible for genomic imprinting is not yet determined, although DNA methylation has been considered the main genetic event for an imprinted mark. In the current study, the authors analyzed the imprinting status and expression levels of the IGFII and H19 genes in 27 cases of Juvenile Nasopharyngeal Angiofibroma (JNA) using RFLPs, RT-PCR, and Southern and Northern Blots. The authors found that four out of eight informative cases (50%) for ApaI/IFGII polymorphism showed biallelic expression of IFGII whereas none of the nine informative cases for the polymorphism showed biallelic expression of the H19 gene. Overexpression of IFGII was observed in 8 out of 22 cases (36.4%), and 7 out of 19 cases (36.8%) showed H19 overexpression. Hypomethylation was found only in the H19 gene in six out of eight cases analyzed. Therefore, our results demonstrate that alterations in the IFGII/H19 imprinted region occur in JNA.

Our aim was to determine the molecular targets involved in the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in a normal murine mammary epithelial cell line, HC11. Among the early response genes analyzed, c-myc, junB, junD, c-jun, c-fos, fosB, fra, as well as max, mad1-4, sin3, only c-jun and fra-2 mRNAs were up-regulated after 1,25(OH)(2)D(3) exposure. Cyclin C was reduced and cyclin A2 and E were slightly enhanced; however, cyclins D1, D3, B1, B2, F, G1, G2, I and H, as well as TGF beta 1, TGF beta 3, T beta RI and T beta RII transcripts were not modulated by 1,25(OH)(2)D(3). Although p27(KIP1) protein content was unchanged, enhancement of p21(WAF1/CIP1) low basal levels in cell extracts and IGFBP-3 abundance on the culture medium was detected after 1,25(OH)(2)D(3) induction. Using differential display analysis, we identified eight down-modulated clones in exposed cells: 26S proteasome non-ATPase subunit Pad1, ubiquitin-conjugating enzyme Ube2i, extracellular proteinase inhibitor Expi or Wdnm1, cytochrome-c oxidase Cox7c, microtubule-associated protein-1 light chain-3 (Map1lc3), nascent-associated complex alpha Naca, transforming acidic coiled-coil Tacc3, stearoyl-CoA desaturase (Scd), keratin 6 alpha, and 1 up-regulated, fork head transcription factor Hfh-1L. Hence, the antiproliferative effect of 1,25(OH)(2)D(3) seems associated to enhancement of c-jun, Fra-2, IGFBP3 and p21(WAF1/CIP1). Decreased Pad1 and Ube2i might account for increased stability of cell cycle inhibitory proteins while reduced Wdnm1, Tacc3 and Scd might be secondary to accumulation of cells in G0/G1 phase.

We have retrospectively analyzed a series of 155 sequential cases of T1N0M0 ductal carcinomas of which 51 tumors had a ductal carcinoma in situ (DCIS) component for correlation between the presence of DCIS and clinicopathological variables, recurrence and patient survival. No correlations between the presence of DCIS and age, menopausal status, size, estrogen or progesterone receptors were found. High-grade infiltrative tumors tended not to present a DCIS component (P = 0.08). Patients with tumors associated with DCIS form a subgroup with few recurrences (P = 0.003) and good survival (P = 0.008). When tumors were classified by size, an association between large tumors (>1.0 cm) and increased recurrence and shortened overall survival was found. The presence of DCIS in this subgroup significantly reduced the relative risk of death.

The present study was undertaken with the aim of evaluating the clinical and anatomopathological findings, emphasizing expression of the protein p53 as possible prognostic markers, in patients with breast sarcoma. p53 immunohistochemical expression was determined in archival paraffin embedded tissue blocks of 30 breast sarcoma patients, (19 fibrosarcomas, nine malignant fibrohistiocytomas and two liposarcomas) treated at the Hospital do Cancer AC Camargo, São Paulo, Brazil from 1955 to 1990. Immunopositivity was present in 50% of the cases. The survival of the patients was compared with the above parameters. Median follow up time was 113 months. The 5 years specific survival rates were 55.1% for patients with a positive expression of p53 contrariwise to 92.3% of specific survival found in p53 negative patients (p = 0.04). Positive expression of p53 was found in 3/4 (75%) of the patients with local recurrence and in 7/9 (77%) of patients with metastatic disease. No significant correlation between survival and clinicopathologic features (age, menopausal status, tumor size, stage and histological type), was found. A slight positive correlation between high grade and poor outcome was observed, 89% of the metastatic cases being classified as high grade (p = 0.02, by one sided Fisher's exact test). When we have compared, independently, survival probability curves between p53 positive/negative expression and each category of clinicopathologic features a worse prognosis was observed when p53 was positive in patients older than 50 years (p = 0.01), in tumors larger than 5 cm (p = 0.02), within the malignant fibrous histiocytoma subtype (p = 0.01) and in tumors classified as high grade (p = 0.07). In conclusion p53 expression seems to be a useful prognostic marker for this type of tumor.

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-beta2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation.

Background: Head and neck squamous cell carcinomas (HNSCC) are known for their invasive behavior. The invasiveness of these tumors requires proteases, some of which as urokinase-type plasminogen activator (uPA), gelatinase B and matrilysin are regulated through AP-1 dependent transcriptional mechanisms. AP-1 consists of several proteins, including those encoded by the proto-oncogenes c-jun and c-fos. The aim of this study was to: first, evaluate the expression levels of matrix metalloproteases (matrilysin and gelatinase B) and uPA mRNAs; second, examine whether these genes might be associated with c-jun and c-fos expression; third, examine the relationship between the expression of these genes and HNSCC clinico-pathological features., Methods: We have analyzed 38 HNSCC primary tumors and matched mucosa tissues for uPA, gelatinase B, matrilysin, c-fos, and c-jun by Northern-blot analysis., Results: uPA, gelatinase B, matrilysin, and c-jun mean levels were statistically higher in the tumors than in the normal adjacent mucosa, whereas no difference was found when c-fos mRNA values were compared, c-jun mRNA expression correlated directly with gelatinase B and matrilysin mRNA levels, but no association with uPA mRNA was observed, c-fos mRNA levels were not associated with the tested proteases, but low levels were determined in tumors from older patients who subsequently developed a 2(nd) tumor. No evidence of correlation between expression of uPA, matrilysin, and c-jun in tumors and clinico-pathological features was found. Gelatinase B mRNA high levels were associated to presence of cervical recurrences., Conclusion: Expression of c-jun seems to be involved in the regulation of gelatinase B and matrilysin being not related to uPA. Lack of association with c-fos may indicate that other fos family members might play a role in the transcriptional activity of the analyzed proteases in HNSCC tumors., (Copyright 2002 John Wiley & Sons, Inc.)

Background: A balance between urokinase-type plasminogen activator (uPA) and its main inhibitor type-1 (PAI-1) appears to be important for cancer invasive behavior. Since uPA/PAI-1 system seems to be regulated by transforming growth factor beta1 (TGFbeta1) in different cell types, our aim was to investigate the relationship between the expression of the three genes and lymph node status in head and neck squamous cell carcinomas (HNSCC) at specific sites., Materials and Methods: uPA, PAI-1, and TGFbeta1 mRNAs were determined by Northern analysis in tumor, and paired normal mucosa samples were obtained from 91 operable HNSCC patients., Results: In oral cavity, excluding tongue, TGFbeta1, PAI-1, and uPA mRNAs values were consistently lower in the normal tissues than in tumors. In larynx tumors, TGFbeta1 expression was increased, but no statistically significant differences were found for uPA or PAI-1 mRNAs as compared with normal tissues. Tongue tumors overexpressed only uPA mRNA, and uPA levels showed significant parallel variations with TGFbeta1 and PAI-1 mRNAs mainly in pN+ tumors. In oral cavity tumors, an inverse correlation between TGFbeta1 and uPA was observed in pN0 subgroup, elevated uPA mRNA was counterbalanced by high PAI-1 mRNA TGFbeta1, and PAI-1 were not coordinately expressed. Correlations between the three markers were not found in larynx. Hypopharynx tumors, all staged as pN+, expressed the lowest TGFbeta1 mRNA mean values., Conclusions: Combined information about TGFbeta1, uPA, and PAI-1 mRNAs may add some clues to the understanding of the pathophysiological role of uPA system in head and neck squamous cell carcinoma., (Copyright 2001 John Wiley & Sons, Inc.)

There are few data evaluating biological markers for men with breast cancer. The purpose of the present study was to analyze the expression of the oncogenes c-erbB-2 and c-myc and of the suppressor gene p53 by immunohistochemical techniques in archival paraffin-embedded tissue blocks of 48 male breast cancer patients, treated at the A.C. Camargo Cancer Hospital, São Paulo, SP, Brazil. The results were compared with clinicopathological prognostic features. Immunopositivity of c-erbB-2, p53 and c-myc was detected in 62.5, 16.7 and 20.8% of the cases analyzed, respectively. Estrogen and progesterone receptors were positive in 75 and 69% of the cases, respectively. Increasing staging was statistically associated with c-erbB-2 (P = 0.04) and weakly related to p53 positivity (P = 0.06). No significant correlation between specific survival rate (determined by the log rank test) and the molecular markers analyzed was found, whereas the number of compromised lymph nodes and advanced TNM (tumor, node, metastasis) staging were associated with diminished survival.

We have identified a novel human gene related to the class 6 semaphorin family of axon guidance molecules, termed human semaphorin 6B or (HSA)SEMA6B. Two splicing variants of this gene were identified by RT-PCR: (HSA)SEMA6B.1 (short isoform) and (HSA)SEMA6B.2 (longer isoform). Computational analysis suggests that these isoforms correspond to putative secreted and transmembranous semaphorins, respectively. The levels of (HSA)SEMA6B expression were evaluated by Northern blot analysis in different tissues and in some pathological and pharmacological conditions. We observed that (HSA)SEMA6B is highly expressed in human brain and at lower levels in a variety of other tissues. Interestingly, the (HSA)SEMA6B transcript was downregulated in two different human glioblastoma cell lines (T98G and A172) upon prolonged treatment with all-trans-retinoic acid, an anti-tumor and differentiation-inducing agent., (Copyright 2001 Academic Press.)

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.

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

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.

Context: Ras gene mutations have been associated to a wide range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras) are structurally related and code for a protein (p21) known to play an important role in the regulation of normal signal transduction and cell growth. The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific., Objectives: To study the occurrence of Ki-ras and Ha-ras mutations. We also studied the relative level of Ha-ras mRNA in 32 of the head and neck tumors., Design: Case series., Setting: University referral unit., Participants: 60 head and neck tumors and in 28 Juvenile Nasopharyngeal Angiofibromas (JNA)., Diagnostic Test: Using PCR-SSCP we examined the occurrence of Ki-ras and Ha-ras mutations. The relative level of Ha-ras mRNA was examined by Northern blot analysis., Results: None of the head and neck tumors or JNA samples showed evidence of mutations within codons 12, 13, 59 and 61 of Ki-ras or Ha-ras genes. However, 17 (53%) of the tumors where gene expression could be examined exhibited increased levels of Ha-ras mRNA compared with the normal tissue derived from the same patient., Conclusions: Our results demonstrate for the first time that mutations of Ki-ras and Ha-ras genes are not associated with the development of JNA and confirm previous reports indicating that activating ras mutations are absent or rarely involved in head and neck tumors from western world patients. Furthermore, our findings suggest that overexpression of Ha-ras, rather than mutations, might be an important factor in the development and progression of head and neck tumors.

HC-11 mammary epithelial cells which originate from midpregnant BALB/c mice are able to differentiate in culture after epidermal (EGF) or basic fibroblast (FGF) growth factor pretreatment followed by lactogenic hormone stimulation (Dexamethasone, Insulin, and Prolactin - DIP). In our study, HC-11 cells exhibited specific vitamin D3 receptors (VDR) determined by Northern analysis or flow cytometry and responded to 10 nM vitamin D3 treatment displaying strong growth inhibition, arrest in G0/G1 phase without evidence of apoptosis, and VDR mRNA reduction, although the percentage of cells expressing VDR protein remained unchanged. In an attempt to verify if there was a correlation between the growth state of the cells and VDR levels, we have examined the effects of growth modulators such as EGF/bFGF and confluency and transformation by Ha-ras. A down-regulation of VDR expression was observed after Ha-ras transformation of HC-11 cells which desensitized the cells to the growth inhibitory effects of vitamin D3. EGF or bFGF decreased VDR in parental cells and EGF antagonized the antiproliferative activity of vitamin D3. As well, transition from proliferating to confluent state significantly reduced VDR levels only in parental cells. DIP-induced HC-11 cell differentiation (monitored by beta-casein transcripts), although leading to cell cycle arrest, increased VDR mRNA content, which seems to be rather related to lactogenic hormone induction than to differentiation itself. In fact, DIP-stimulated HC-11 cells in the absence of EGF pretreatment, or DIP-treated HC-11ras cultures, also displayed up-regulated VDR level even in the absence of differentiation. Concluding, mammary VDR levels might be regulated by growth modulating agents, by physiological conditions of the gland, and by the ras-mediated malignant transformation.

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.

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.

Eosinophils accumulate into the uterus of ovariectomized rats, after treatment with estradiol (E2). We have investigated whether this feature is related to interactions of eosinophils with uterine extracellular matrix proteins: laminin (LM) and fibronectin (FN). Eosinophils isolated from the peritoneal cavity of ovariectomized rats displayed estrogen receptors measured at both binding activity and mRNA levels. An increased number of laminin binding sites, calculated by Scatchard analysis using iodinated LM was determined in E2-treated eosinophils (70,100 +/- 28,000 sites/cell vs 21,000 +/- 5,000 sites/cell in controls). Eo binding to 125I-LM- was inhibited by the E8-LM fragment. Estradiol up-regulated the expression in eosinophils of alpha 6 and beta 2 integrin subunits evaluated by flow-cytometry as well as by alpha 6 mRNA expression. After E2 treatment, eosinophils showed higher adhesiveness to LM-coated dishes (10 +/- 2 vs 56 +/- 3%) which was inhibited by monoclonal antibodies against alpha 6, beta 1 and beta 2 integrins and by the steroid antagonist tamoxifen. These monoclonal antibodies also blocked the attachment of stimulated eosinophils to uterine cryostat sections obtained from spayed rats previously treated with estradiol. We did not detect any apparent influence of E2 on basal eosinophil adherence or binding to FN although alpha 4 and alpha 5 integrin subunits were expressed in eosinophils. Expression of laminin and merosin in the uterus was determined immunohistochemically. Our results suggest that integrin-laminin interactions may contribute to the preferential eosinophil recruitment in vivo.

We have investigated the effects of laminin, on the plasminogen-activator system of MCF-7 breast-carcinoma cells. MCF-7 cells were incubated on plastic or laminin-coated wells, and medium and cell lysate aliquots were assayed for tissue-type (tPA) and urokinase-type plasminogen activator (uPA) by a chromogenic assay in combination with anti-uPA antibodies. Cells cultured on laminin displayed a 5-fold increase in tPA activity and a 2-fold decrease in uPA activity relative to cells on plastic. These effects could be mimicked by laminin fragment P1 but not by collagen I or fibronectin. tPA activity of cells treated with estradiol (10 nM) was 3-fold higher, that of cells on laminin treated with estradiol was 15-fold higher, than that of control. Northern-blot analysis showed that tPA mRNA levels were up-regulated by estradiol and laminin, whereas PAI-1 mRNA levels were down-regulated by laminin and not affected by E2. Concomitant treatment with laminin and estradiol, decreased PAI-1 mRNA and increased tPA mRNA levels, accounting for the synergistic increase in tPA activity. Laminin exerted only a modest (approx. 2-fold) inhibitory effect on uPA mRNA levels. In the breast-carcinoma cell line MDA-MB-231, down-regulation of PAI-1 and uPA mRNA by laminin was not observed. Adhesion assays indicated that alpha2beta1 is the predominant receptor for laminin in MCF-7 cells. MDA-MB-231 cells expressed alpha2 (54%) but this integrin is not used as a laminin receptor. These results support a role for alpha2beta1 in mediating interactions of MCF-7 with LN.

MCF-7 (estrogen receptor positive--ER+) and MDA-MB-231 (estrogen receptor negative--ER-) are human breast cancer cell lines which express functional thyroid hormone receptors (c-erb A alpha1 and c-erb beta1) as indicated by stimulation of mitochondrial alpha-glycerophosphate dehydrogenase. In MCF-7, mimicking E2, T3 stimulated growth in a dose-dependent (10(10) M - 10(-8) M) manner, induced the expression of progesterone receptor and growth factor TGFalpha mRNAs and inhibited that of TGFbeta mRNA; T3 also increased progesterone binding and LDH5 isozyme activities. None of these effects were observed in (ER-) MDA-MB-231 cells. 10(-6) M tamoxifen (TAM) reverted growth stimulation, suppressed progesterone receptor and TGFalpha mRNA induction and restored TGFbeta mRNA to control levels in T3-treated MCF-7 cells. That T3 is acting in MCF-7 cells via its binding to ER is suggested by the immunoprecipitation of pre-bound 125I-T3 from MCF-7 nuclear extracts by an ER-specific monoclonal antibody and by the displacement of 3H-estradiol binding to ER by radioinert T3.

Biopsies from 25 juvenile nasopharyngeal angiofibromas (JNAs) and respective normal inferior turbinates were examined and compared. The expression patterns of the messenger RNAs (mRNAs) for various growth factors possibly involved in the growth of mesenchymal cells, as well as angiogenesis and fibrosis, were also compared. These growth factors included insulin-like growth factor II (IGF-II), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factors-beta1 (TGF-beta1) and platelet-derived growth factors (PDGF-A and PDGF-B). Quantification of mRNA coding for proto-oncogenes and suppressor genes related to proliferation (i.e., c-myc, c-fos, p53) was also undertaken. Tumor and turbinates expressed similar levels of bFGF, VEGF, TGF-beta1, c-myc, c-fos, and PDGF-A mRNAs. The presence of TGF-beta1 protein was confirmed by immunohistochemistry in several structures that characterize the lesions of JNA, which suggests that TGF-beta1 may play a role in the development of the fibrous component of this tumor. PDGF-B and p53 were overexpressed (i.e., twice the mean level found in turbinates) in 50% and 32% of JNAs, respectively but there was no statistical significance when compared with controls. Statistically significant increased expression of IGF-II mRNA was observed in JNA (P = .04). IGF-II mRNA levels were correlated to p53 (P = .05) and PDGF-B (P = .034), indicating a possible synergistic action of such factors in JNA. The results of this study suggest that IGF-II might be a potential growth regulator of nasopharyngeal angiofibromas.