Response Of Cancer Cell Lines Research Articles

Migration of Breast Cancer Cell Lines in Response to Pulmonary Laminin 332; Implications for Metastasis

In order to determine whether lung tissue secretes a motility factor for breast cancer, minced fresh human pulmonary tissue was co‐cultured with MCF‐7 breast carcinoma cells. Dose‐dependent MCF‐7 migration was observed, consistent with a motility response to a factor secreted by the lung tissue. Pulmonary epithelial cells were isolated and cultured in defined medium. Concentrated conditioned lung culture medium induced motility in the breast carcinoma cell lines MDA‐MB‐231, SKBR3, and BT‐20 using a Boyden chamber assay. For MCF‐7, the motility was dose dependent by a scattering assay, when the concentrate was either in a soluble form or after deposition on tissue culture plastic. The deposited material was analyzed by mass spectrometry, revealing the α3 and β3 chains of the motility factor laminin 332 (LN332). Blocking antibodies to LN332 and to the LN332 receptor for motility, α3β1 integrin, inhibited motility to the same extent as the inhibition of motility that occurred when these antibodies were applied to MCF‐7 cells incubated with purified LN332. Finally, LN332 in normal lung was confirmed by immunohistochemistry. These experiments together showed that pulmonary LN332 induced motility in MCF‐7. These findings provide a mechanism for the migration of metastatic tumor cells from their arrest in the pulmonary vasculature into the surrounding tissue, allowing their establishment as a metastatic colony.

PTMScan Direct: Identification and Quantification of Peptides from Critical Signaling Proteins by Immunoaffinity Enrichment Coupled with LC-MS/MS

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

5‐Aza‐2'‐deoxycytidine reactivates gene expression via degradation of pRb pocket proteins

Not only does 5-aza-2'-deoxycytidine (5-aza-CdR) induce the reexpression of silenced genes through the demethylation of CpG islands, but it increases the expression of unmethylated genes. However, the mechanism by which 5-aza-CdR activates the expression of genes is not completely understood. Here, we report that the pRb pocket proteins pRb, p107, and p130 were degraded in various cancer cell lines in response to 5-aza-CdR treatment, and this effect was dependent on the proteasome pathway. Mouse double minute 2 (MDM2) played a critical role in this 5-aza-CdR-induced degradation of pRb. Furthermore, PP2A phosphatase-induced MDM2 dephosphorylation at S260 was found to be essential for MDM2 binding to pRb in the presence of 5-aza-CdR. pRb degradation resulted in the significant reexpression of several genes, including methylated CDKN2A, RASFF1A, and unmethylated CDKN2D. Finally, knockdown of pRb pocket proteins by either RNAi or 5-aza-CdR treatment induced a significant decrease in the recruitment of SUV39H1 and an increase in the enrichment of KDM3B and KDM4A to histones around the promoter of RASFF1A and thus reduced H3K9 di- and trimethylation, by which RASFF1A expression is activated. Our data reveal a novel mechanism by which 5-aza-CdR induces the expression of both methylated and unmethylated genes by degrading pRb pocket proteins.

Role of Src in breast cancer cell migration and invasion in a breast cell/bone-derived cell microenvironment

The preferential metastasis of breast cancer cells to bone comprises a complex set of events including homing and preferential growth, which may require unique factors produced by bone or other cells in the immediate microenvironment. In this study, an in vitro co-culture system composed of bone mesenchymal stem cells and breast cancer cell lines is used to examine the role of Src kinase on breast cancer cell migration and invasion in the presence of bone-derived cells. This research shows that Src kinase activity in breast cancer cell lines with either high or low levels of endogenous Src activity is increased by bone-derived cell-conditioned medium but not HS68 fibroblast-conditioned medium. Breast cancer cells exhibit enhanced migration in co-culture with bone-derived cells but not HS68 fibroblasts or no co-cultured cells. Inhibition of Src kinase activity using the inhibitors PP2 or saracatinib or using siRNA abrogates the preferential migration of the breast cancer cell lines in response to bone-derived cells. Inhibition of Src activity with saracatinib does not have any significant effect on breast cancer cell invasion in the presence of bone-derived cells. Factors are identified that are produced preferentially by bone-derived cells over HS68 cells that may impact breast cancer cell behavior. This research implicates Src kinase as an important effector of bone-derived cell signals on breast cancer cell migration.

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKβ. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity

Autophagy is characterized by the sequestration of bulk cytoplasm, including damaged proteins and organelles, and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity, the mechanism is not yet clear. Here we report that cytosolic FoxO1, a forkhead O family protein, is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation, but this process was independent of the transcriptional activity of FoxO1. In response to stress, FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2), a NAD(+)-dependent histone deacetylase, and the acetylated FoxO1 bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy.

Abstract C150: SOX9 modulates Wnt/beta-catenin singal in cancer cells by regulating TCF4 and LRP6 expression

Abstract Introduction: Cancers frequently exploit developmental genes/pathways to sustain their uncontrolled growth or invasion. SOX9, an important developmental transcription factor is one of the signature genes of the basal-like breast cancers. SOX9 over-expression has been demonstrated in various cancers and is associated with a poor prognosis. In prostate cancer, SOX9 supports tumor proliferation, angiogenesis and local invasion in xenografts. The role of SOX9 as a biomarker and molecular mechanisms of its biological functions were further explored in this study. Methods: Immunohistochmical analysis of SOX9 protein expression was performed on a tissue microarray of 129 breast cancer samples. Gene profiles of the breast cancer samples were examined by ANOVA analysis to identify genes that are expressed at significantly higher level among SOX9-high protein expressing samples. SOX9 regulated genes were validated by their corresponding changes in expression levels to SOX9 silencing in cancer cell lines. The effect of SOX9 silencing on cancer progression was examined in the MCF10A DCIS.com xenografts. Results: SOX9 protein expression was highly represented in the basal-like breast cancers and was associated with poor tumor grades. Among the genes correlated with higher SOX9 expression were two major components of the canonical Wnt/ -catenin signal pathway, ie TCF4 and LRP6. These genes were up- or down-regulated in various cancer cell lines in response to SOX9 overexpression or silencing. More importantly, the Wnt3a induced -catenin activity was significantly reduced when SOX9 protein was specifically down-regulated. SOX9 down-regulation in MCF10A DCIS xenografts lead to a squamous differentiation and a block in the progression from DCIS to invasive carcinoma. Conclusion: This study identified a novel function of SOX9 in modulating Wnt/β-catenin signaling by regulating TCF4 and LRP6 expression. SOX9 enhanced Wnt/β-catenin activity in the absence of APC or β-catenin mutations may play an important role in determining tumor subtype, maintaining tumor stem cells or supporting tumor growth or progression. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C150.

P68 Proteomic changes in colorectal cancer cell lines in response to sorafenib treatment

P68 Proteomic changes in colorectal cancer cell lines in response to sorafenib treatment

Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of α6β1 integrin

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial–mesenchymal interconversions leading to the disruption of cell–cell contact and modulation of cell–extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in α2, α6 and β1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of α6 and β1 integrin subunits. Neutralizing antibodies against α6 and β1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of α6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475–85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and α6β1 integrin-mediated signalling in ovarian carcinomas.

Blocked Autophagy Sensitizes Resistant Carcinoma Cells to Radiation Therapy

Autophagy or "self eating" is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to gamma-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific target-siRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells.

Radiotherapy in Lung Adenocarcinoma with Brain Metastases: Effects of Activating Epidermal Growth Factor Receptor Mutations on Clinical Response

Whole-brain radiation therapy (WBRT) has been applied to inoperable brain metastases in lung adenocarcinoma. Recently, an in vitro study showed reduced clonogenic survival of mutant epidermal growth factor receptor (EGFR) lung cancer cell lines in response to ionizing radiation compared with that of the wild type. To elucidate the role of EGFR mutations in radiation treatment, we evaluated the clinical response to WBRT and survival of lung adenocarcinoma patients with brain metastases. This was a retrospective analysis of 63 patients with brain metastases from lung adenocarcinoma who were treated with WBRT. Demographic data, EGFR mutation status, response to WBRT, and survival data were collected. Clinical response was assessed 1 month after the start of WBRT. Univariate and logistic regression models were used to test potential predictive factors associated with clinical response. Log-rank test and Cox regression were analyzed to identify factors that affected survival. Clinical response to WBRT was observed in 29 patients (46%), with 34 nonresponder patients (54%). Patients with EGFR mutations had higher response rates to WBRT compared with those with the wild-type (54% versus 24%; P = 0.045). Both the administration of EGFR tyrosine kinase inhibitor (P = 0.034) and EGFR mutation (P = 0.029) were independently associated with response to WBRT. In Cox regression analysis, WBRT responder (P = 0.010) and absence of extracranial metastases (P = 0.002) were associated with better survival. Both the EGFR mutations and the administration of EGFR TKI during WBRT were independent predictors of response to WBRT in brain metastases of lung adenocarcinoma.

Epithelial–mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: An exception to the norm

Cancer that arises from the ovarian surface epithelium (OSE) accounts for approximately 90% of human ovarian cancer, and is the fourth leading cause of cancer-related deaths among women in developed countries. The pathophysiology of epithelial ovarian cancer is still unclear because of the poor understanding of the complex nature of its development and the unusual mechanism(s) of disease progression. Recent studies have reported epithelial-mesenchymal transition (EMT) in cultured OSE and ovarian cancer cell lines in response to various stimuli, but our understanding of the importance of these observations for normal ovarian physiology and cancer progression is not well established. This review highlights the current literature on EMT-associated events in normal OSE and ovarian cancer cell lines, and discusses its implication for normal ovarian function as well as acquisition of neoplastic phenotypes. The pathological changes in OSE in response to EMT during neoplastic transformation and the contribution of hormones, growth factors, and cytokines that initiate and drive EMT to sustain normal ovarian function, as well as cancer development and progression are also discussed. Finally, emphasis is placed on the clinical implications of EMT and potential therapeutic opportunities that may arise from these observations have been proposed.

SiRNA‐mediated type 1 insulin‐like growth factor receptor silencing induces chemosensitization of a human liver cancer cell line with mutant P53

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.

Influence of Genistein Isoflavone on Matrix Metalloproteinase-2 Expression in Prostate Cancer Cells

We investigated the expression of matrix metalloproteinase (MMP)-2 in human LNCaP and PC3 prostate cancer cell lines in response to genistein exposure. Initially we studied the phytosensitivity of the cells to genistein using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to determine percentage cell viability/inhibition and the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labeling apoptosis assay to assess the type of cell death. The results revealed that genistein inhibited growth and proliferation in both PC3 (hormone-dependent) and LNCaP (hormone-independent) prostate cancer cell lines, that there was no significant difference in sensitivity to genistein between PC3 and LNCaP cells, and that the effect of genistein on the cells was dose- and time-dependent. The results also revealed that inhibition of cell growth in both PC3 and LNCaP cells was predominantly due to apoptotic cell death. These results were consistent with data in previous studies. This was followed by determination of the MMP-2 profile in response to genistein treatment. The results indicated a significant dose- and time-dependent inhibition of MMP-2 expression levels in both cells, with a highly significant negative correlation between MMP-2 levels and concentration of genistein. This is of phytotherapeutic significance in view of the pivotal role of MMP-2 expression in the pathogenesis of prostate cancer. Increasing expression of MMPs has been identified in many human cancers, including prostate cancer. Our findings indicate that genistein could be a potent therapeutic inhibitor of MMP-2 in line with current concepts of targeted treatment.

The effects of the inhibition of inducible nitric oxide synthase on angiogenesis of epithelial ovarian cancer

The release of nitric oxide by tumor cells, through the stimulation of inducible nitric oxide synthase expression, may play a critical role in ovarian cancer progression. In this study we have sought to determine the effects of inhibiting inducible nitric oxide synthase on angiogenesis that was induced by 2 ovarian cancer cell lines, SKOV and MDAH2774. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay techniques were used to determine the expression levels of inducible nitric oxide synthase and vascular endothelial growth factor in the ovarian cancer cell lines in response to treatments with L-NAME, an inhibitor of nitric oxide synthase, and SNAP, and nitric oxide donor. Ovarian cancer-induced angiogenesis was assessed in vitro with an established assay that is based on the ability of human umbilical vein endothelial cells to form a tubular network in response to angiogenic agents. SKOV and MDAH2774 cell lines exhibited over-expression of inducible nitric oxide synthase and have high baseline nitric oxide levels. This was associated with high levels of vascular endothelial growth factor production and angiogenesis induction. Treatment of the ovarian cancer cell lines with L-NAME significantly reduced vascular endothelial growth factor levels production and completely inhibited angiogenesis. In contrast, treatment with SNAP significantly increased vascular endothelial growth factor levels and increased angiogenesis in both cell lines. Our data suggest that the inhibition of inducible nitric oxide synthase may form a basis for a novel therapeutic treatment option for ovarian cancer therapy.

Small interfering RNA targeting survivin sensitizes lung cancer cell with mutant p53 to adriamycin

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is specifically overexpressed in cancer tissues. p53 is one of the tumor suppressor genes; its induction in response to DNA damage causes apoptosis and correlates with drug sensitivity. To investigate the possible regulation of survivin by p53, we examined the level of survivin expression in lung cancer cell lines in response to adriamycin. Levels of survivin mRNA and protein in cell lines with wild-type p53 decreased dramatically after p53 induction, but no such reduction of survivin was observed in cell lines with mutated or null p53. Inhibition of wild-type p53 in A549 cells by small interfering (si) RNA significantly upregulated the expression of survivin. Survivin inhibition by siRNA in PC9 cells with mutated p53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of survivin, we depressed survivin expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 hr incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting survivin, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting survivin. Our results demonstrate that survivin is downregulated by p53, and that siRNA targeting of survivin increases cell sensitivity to adriamycin and promotes apoptosis. siRNA targeting of survivin could be potentially useful for increasing sensitivity to anticancer drugs, especially in drug-resistant cells with mutated p53.

Different effect of beta-carotene on proliferation of prostate cancer cells

It was shown that high doses of beta-carotene (>30 microM) decrease proliferation of prostate cancer cells in vitro. However, it is rather doubtful whether such concentration of beta-carotene is really accessible at cellular level. We studied the effect of 3 and 10 microM beta-carotene on proliferation and gene expression in LNCaP and PC-3 prostate cancer cell lines. Beta-carotene--more efficiently absorbed from medium by androgen-sensitive LNCaP cells--increased proliferation of LNCaP cells whereas it had weaker effect on PC-3 cells. Initial global analysis of expression of genes in both cell lines treated with 10 microM beta-carotene (Affymetrix HG-U133A) showed remarkable differences in number of responsive genes. Their recognition allows for conclusion that differences between prostate cancer cell lines in response to beta-carotene treatment are due to various androgen sensitivities of LNCaP and PC-3 cells. Detailed analysis of expression of selected genes in beta-carotene treated LNCaP cells at the level of mRNA and protein indicated that the observed increase of proliferation could have been the result of slight induction of a few genes affecting proliferation (c-myc, c-jun) and apoptosis (bcl-2) with no significant effect on major cell cycle control genes (cdk2, RB, E2F-1).

Nonlinear Dose-Effect Relationship of Different Parameters in Cancer Cell Lines

The dose-effect curves of most parameters in cancer cell lines in response to x-irradiation or toxic agents are usually nonlinear. Such a nature is demonstrated in this article with cell lines of the immune system. However, the shape of the nonlinear dose-effect curves was found to be different for lymphocytes (EL-4) and macrophages (J774A.1). The mechanisms and implications are briefly discussed.

MUC1 expression in human prostate cancer cell lines and primary tumors.

MUC1 expression was evaluated in normal prostate epithelial cells (PrEC), and prostate cancer cell lines in response to dihydrotestosterone (DHT), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment. Expression of MUC1 core protein was stimulated in PrEC and PC-3 cells after cytokine treatment, but was highly and constitutively expressed by DU-145 cells. MUC1 was not expressed by LNCaP, C4-2 or C4-2B cells under any condition. DHT alone or in combination with cytokines had no effect on MUC1 expression in any cell line tested. Using antibodies capable of detecting all isoforms of MUC1 core protein independent of their glycosylation state, immunohistochemical staining of tissue microarrays containing both nontumor and tumor tissue revealed that only 17% of tumor tissues and 41% of nontumor tissues stained positively for MUC1. Staining patterns in tumor tissue varied from focal apical staining to diffuse cytoplasmic staining. Neither the presence of MUC1 core protein nor its subcellular distribution correlated with Gleason grade. These data indicate that MUC1 is a poor marker of prostate cancer progression. Furthermore, IFN-gamma and TNF-alpha strongly induce MUC1 expression in both normal prostate epithelia and certain prostate tumor cell lines and may exacerbate pathologies associated with MUC1-positive prostate cancers.

Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation.

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the alpha(2), alpha(5) and beta(1) integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the beta(1) integrin subunit. Antibodies to alpha(3) integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with collagen. It seems that alpha(3) subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.