Biomedical subjects

Andra R Frost

Publications and source records attributed to Andra R Frost.

At least 19 recordsLinked to original sources

PubMed2006

Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients.

PURPOSE: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. EXPERIMENTAL DESIGN: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. RESULTS: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P=0.006) or HER2 positive (P=0.039) and <50 years old at diagnosis (P=0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P=0.008) or PR (P=0.029) or HER2 overexpression (P=0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. CONCLUSIONS: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.

Adult↗

PubMed2006

O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptors.

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.

Animals↗

PubMed2006

Osteopontin knockdown suppresses tumorigenicity of human metastatic breast carcinoma, MDA-MB-435.

Elevated expression of osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with many transformed cell lines. Various studies suggest that OPN may contribute to tumor progression as well as metastasis in multiple tumor types. High levels of OPN have been reported in patients with metastatic cancers, including breast. We found that the expression of OPN corroborates with the aggressive phenotype of the breast cancer cells i.e. the expression of OPN is acquired as the breast cancer cells become more aggressive. To assess the role(s) of OPN in breast carcinoma, expression of endogenous OPN was knocked down in metastatic MDA-MB-435 human breast carcinoma cells using RNA interference. We targeted multiple regions of the OPN transcript for RNA interference, along with 'scrambled' and 'non-targeting siRNA pool' controls to distinguish between target-specific and potential off-target effects including interferon-response gene (PeIF2-alpha) induction. The OPN knockdown by shRNA suppressed tumor take in immunocompromised mice. The 'silenced' cells also showed significantly lower invasion and migration in modified Boyden chamber assays and reduced ability to grow in soft agar. Thus, in addition to the widely reported roles of OPN in late stages of tumor progression, these results provide functional evidence that OPN contributes to breast tumor growth as well.

Animals↗

PubMed2006

Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer.

The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to noncancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.

Base Sequence↗

PubMed2006

Kinetics of metastatic breast cancer cell trafficking in bone.

PURPOSE: In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention. EXPERIMENTAL DESIGN: MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1 hour to 6 weeks. RESULTS: Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to approximately 60% of control). CONCLUSIONS: Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.

Animals↗

PubMed2006

Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Animals↗

PubMed2005

KLF4 and PCNA identify stages of tumor initiation in a conditional model of cutaneous squamous epithelial neoplasia.

KLF4 is induced upon growth-arrest in vitro and during epithelial maturation in vivo, and is essential for proper cell fate specification of post-mitotic cells. In spite of a normal role in post-mitotic cells, expression is upregulated and constitutive in certain tumor types. KLF4 functions as an oncogene in vitro, and enforced expression in basal cells of mouse skin rapidly induces lesions similar to hyperplasia, dysplasia and squamous cell carcinoma (SCC). Here we used conditional expression to characterize early steps in KLF4-mediated tumor initiation. In contrast to SCC-like lesions that result when using a conditional, keratin 14 promoter-dependent strategy, lower conditional expression achieved using a MMTV promoter induced only epidermal cycling within morphologically normal skin, a process we termed occult cell turnover. Surprisingly, KLF4-induced hyperplastic lesions showed increased transgene-derived mRNA and protein in maturing, PCNA-negative cells, a property of endogenous KLF4. In contrast, hyperplastic lesions induced by GLI1, a control, showed uniform transgene expression. In KLF4-induced dysplasia and SCC the complementarity of KLF4 and PCNA was replaced by concordance of the two proteins. These studies show that KLF4 transcripts are normally suppressed in cycling cells in a promoter-independent fashion, consistent with a post-transcriptional control, and reveal loss of this control in the transition from hyperplasia to dysplasia. Like the mouse tumors, human cutaneous SCCs and adjacent dysplasias frequently showed maturation-independence of KLF4, with co-expression of KLF4 and PCNA. A smaller subset of human SCCs showed complementarity of KLF4 and PCNA, similar to hyperplastic mouse skin. The results identify parallels between a mouse model and human primary tumors, and show that successive increases of KLF4 in the nuclei of basal keratinocytes leads to occult cell turnover followed by hyperplasia, dysplasia, and invasive SCC.

Animals↗

PubMed2005

Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility.

Colon adenocarcinomas are known to express elevated levels of alpha2-6 sialylation and increased activity of ST6Gal-I, the Golgi glycosyltransferase that creates alpha2-6 linkages. Elevated ST6Gal-I positively correlates with metastasis and poor survival, and therefore ST6Gal-I-mediated hypersialylation likely plays a role in colorectal tumor invasion. Previously we found that oncogenic ras (present in roughly 50% of colon adenocarcinomas) up-regulates ST6Gal-I and, in turn, increases sialylation of beta1 integrin adhesion receptors in colon epithelial cells. However, we wanted to know if this pattern held true in vivo and, if so, how beta1 hypersialylation might contribute to colon tumor progression. In the present study, we find that beta1 integrins from colon adenocarcinomas consistently carry higher levels of alpha2-6 sialic acid. To explore the effects of increased alpha2-6 sialylation on beta1-integrin function, we stably expressed ST6Gal-I in a colon epithelial cell line lacking endogenous ST6Gal-I. ST6Gal-I expressors (with alpha2-6 sialylated beta1 integrins) exhibited up-regulated attachment to collagen I and laminin and increased haptotactic migration toward collagen I, relative to parental cells (with completely unsialylated beta1 integrins). Blockade of ST6Gal-I expression with short interfering RNA reversed collagen binding back to the level of ST6Gal-I nonexpressors, confirming that alpha2-6 sialylation regulates beta1 integrin function. Finally, we show that beta1 integrins from ST6Gal-I expressors have increased association with talin, a marker for integrin activation. Collectively, these findings suggest that beta1 hypersialylation may augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration.

Adenocarcinoma↗

PubMed2005

Identification and immunohistochemical localization of Sulfotransferase 2B1b (SULT2B1b) in human lung.

Sulfotransferase 2B1b (SULT2B1b) is a member of SULT 2 gene family. SULT2B1a and SULT2B1b are transcribed from the same gene using different transcriptional start sites and contain different first exons as the result of alternative splicing. SULT2B1a and SULT2B1b are 350 and 365 AA in length, respectively. Northern blot analysis and SULT2B1 isoform specific RT-PCR detected only SULT2B1b message in human lung tissue. Immunoblot analysis of human lung tissue with a specific rabbit anti-SULT2B1 polyclonal antibody detected only SULT2B1b immunoreactive protein in human lung cytosol. Immunoprecipitation and MALDI mass spectroscopic analysis verified that the immunoreactive protein was SULT2B1b. Immunohistochemical localization of SULT2B1b in human tissues showed expression in the cytoplasm of ciliated columnar or cuboidal epithelial cells in terminal bronchia. No staining was observed in alveolar cells. SULT2B1b is selective for the sulfation of 3beta-hydroxysteroids such as dehydroepiandrosterone and pregnenolone as well as cholesterol. The presence of SULT2B1b in lung tissues suggests a role in the regulation of local steroid hormone synthesis and metabolism.

Bronchi↗

PubMed2005

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.

Animals↗

PubMed2005

Cholangiocyte endothelin 1 and transforming growth factor beta1 production in rat experimental hepatopulmonary syndrome.

BACKGROUND & AIMS: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor beta1 regulates endothelin 1 production. METHODS: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor beta1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. RESULTS: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor beta1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor beta1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor beta1 and phosphorylated Smad2 levels. Transforming growth factor beta1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. CONCLUSIONS: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor beta1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome.

Animals↗

PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Biomarkers↗

PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Coloring Agents↗

PubMed2004

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.

BACKGROUND: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. METHODS: NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. RESULTS: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. CONCLUSION: Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth.

Breast↗

PubMed2004

Different subcellular localization of sulphotransferase 2B1b in human placenta and prostate.

The human hydroxysteroid SULT (sulphotransferase) 2B1 subfamily consists of two isoforms, SULT2B1a and SULT2B1b. These two isoenzymes are transcribed from the same gene by alternative splicing of their first exons and share 94% amino acid sequence identity. The SULT2B1 isoforms are highly selective for the sulphation of 3beta-hydroxysteroids. Immunoblot analysis of SULT2B1 expression in several human tissues indicates the presence of only SULT2B1b protein. Immunoreactive SULT2B1b protein was detected in human prostate, skin, placenta and lung tissue. SULT2B1b mRNA expression was detected in RNA isolated from term placenta, normal prostate, prostate carcinoma, benign prostate hyperplasia, LNCaP prostate cancer cells, breast cancer specimens and MCF-7 breast cancer cells. Immunohistochemical localization of SULT2B1b, in terms placental and prostate tissues, detected it in nuclei of placental syncytiotrophoblasts and cytoplasm of epithelial cells in prostate tissues. Immunoreactive and catalytically active SULT2B1b was identified in nuclei isolated from term human placenta. Also SULT2B1b was capable of translocating to nuclei in BeWo placental cells after stable transfection and differentiation. In contrast, immunohistochemical analysis of human prostate showed only cytosolic localization of SULT2B1b in the basal and luminal prostate epithelial cells. SULT2B1b was not detected in isolated nuclei from LNCaP prostate cancer cells but was present in the cytosolic fraction. Differential subcellular localization of SULT2B1b in prostate and placenta suggests that SULT2B1b may be differentially regulated and have different physiological functions in these two hormonally responsive human tissues.

Blotting, Western↗

PubMed2004

Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer.

PURPOSE: The Krüppel-like transcription factor KLF4/GKLF induces both malignant transformation and a slow-growth phenotype in vitro. Although KLF4 expression is increased in most cases of breast cancer, it was unknown whether these cases represent a distinct subtype with a different clinical outcome. EXPERIMENTAL DESIGN: We examined expression of KLF4 by immunostaining 146 cases of human primary infiltrating ductal carcinoma of the breast. Staining patterns were correlated with clinical outcome and with established prognostic factors. RESULTS: Subcellular localization exhibited case-to-case variation. Tumors with high nuclear staining and low cytoplasmic staining were termed type 1. For patients with early-stage disease (i.e., stage I or IIA), type 1 staining was associated with eventual death because of breast cancer (hazard ratio, 2.8; 95% confidence interval, 1.23-6.58; P = 0.011). The association was stronger in patients with early-stage cancer and small primary tumors (i.e., < or =2.0 cm in diameter; hazard ratio, 4.3; 95% confidence interval, 1.75-10.62; P < 0.001). For patients with early-stage disease, multivariate analysis indicated that type 1 staining was independently associated with outcome (adjusted hazard ratio 2.6; 95% confidence interval, 1.10-6.05; P = 0.029). Type 1 staining was also associated with high histological grade (P = 0.032), increased expression of Ki67 (P = 0.016), and reduced expression of BCL2 (P = 0.032). In vitro, KLF4 was localized within the nucleus of transformed RK3E epithelial cells, consistent with a nuclear function of this transcription factor during induction of malignant transformation. CONCLUSIONS: The results suggest that localization of KLF4 in the nucleus of breast cancer cells is a prognostic factor and identify KLF4 as a marker of an aggressive phenotype in early-stage infiltrating ductal carcinoma.

Breast Neoplasms↗

PubMed2003

Detecting the expression of human endogenous retrovirus E envelope transcripts in human prostate adenocarcinoma.

BACKGROUND: The expression of human endogenous retrovirus (HERV) mRNA and proteins was associated recently with diseases that include human malignancies. The authors report that, in the current study, transcripts encoding the envelope region of an HERV family, HERV-E, were expressed in human prostate carcinoma. METHODS: RNA was isolated from various prostate tissues and was tested for the expression of various HERV envelope (env) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, RNA in situ hybridization (ISH), and Northern blot analysis. Variants of HERV that appeared in prostate carcinoma tissues were sequenced, and HERV-E was expressed in prokaryotic and eukaryotic systems. RESULTS: In the current study, the authors found that the mRNA of the env gene of one particular family of HERVs, HERV-E, was expressed in some prostate carcinoma tissues (38.8% positive; n = 49 specimens) but not in normal prostate tissues using RT-PCR, RNA ISH, and Northern blot assays. The expression of HERV-E transcripts in prostate tumor epithelial cells was confirmed further by ISH using an HERV-E specific antisense probe. Approximately 50% of the cDNA of HERV-E obtained from prostate carcinoma specimens contained no stop codon and expressed proteins in prokaryotic or eukaryotic expression systems. Furthermore, the expression of both HERV-E and ERV3 (another class of HERV) was detected in the same prostate carcinoma tissues. CONCLUSIONS: The expression and distribution of multiple HERV-E endogenous retroviral elements in prostate carcinoma, but not in normal control specimens, suggests that they may serve as novel tumor markers for the early diagnosis and immunotherapy of patients with prostate carcinoma.

Blotting, Northern↗

PubMed2003

LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer.

Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G2/M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasis-associated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).

Actin Depolymerizing Factors↗