Biomedical subjects

Jürgen Dittmer

Publications and source records attributed to Jürgen Dittmer.

14 recordsLinked to original sources

PubMed2006

Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells.

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.

Biopsy↗

PubMed2006

Beta-actin is not a reliable loading control in Western blot analysis.

Beta-actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.

Actins↗

PubMed2006

Ezrin expression is related to poor prognosis in FIGO stage I endometrioid carcinomas.

As a cortical cytoskeletal protein, ezrin adapts the cytoplasmic tail of CD44 to the actin-based cytoskeleton and is functionally involved in migration and adhesion that are prerequisites for metastasis. To assess the importance of ezrin and its associated protein osteopontin for the progression of endometrioid carcinoma in FIGO stage I, we analyzed paraffin-embedded tissue from 164 patients by immunohistochemistry and correlated these data with clinicopathological parameters. Ezrin was expressed in normal proliferating endometrial glands, as was confirmed by quantitative PCR and immunohistochemistry. In endometrioid carcinoma, enhanced ezrin expression correlated with a reduced overall survival in univariate analysis (P = 0.041). In contrast, no significant correlation was found for osteopontin. In multivariate survival analysis, among FIGO grade 3 and age, ezrin was still found to be an independent risk factor (relative risk 2.2, confidence interval 1.0-5.4, P = 0.047). Hence, elevated ezrin expression is a new independent prognostic marker in FIGO stage I endometrioid carcinoma, and thus provides further evidence for an important role of ezrin in tumor progression.

Aged↗

PubMed2006

Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma.

The majority of patients diagnosed with ovarian carcinoma are classified as being in advanced stage of disease. In a situation of cancer spread throughout the abdominal cavity, a successful curative treatment is difficult to achieve. Therefore, preventing binding of tumor cells to the mesothelium is crucial for patients' outcome. One important mechanism is the interaction between hyaluronic acid and the CD44 receptor with its submembrane linking complex. This consists of ezrin, radixin, and moesin and connects the CD44 receptor with the cytoskeleton. To assess the role of ezrin and moesinfor ovarian carcinoma progression, we analyzed ovarian carcinoma samples from 105 patients for expression of ezrin and moesin by immunohistochemistry and correlated these data with several clinicopathological parameters. To elucidate the functional importance of ezrin and moesin, their expression was inhibited in SKOV-3 cells by RNA interference. Ezrin and moesin were strongly expressed in 49 and 48% of ovarian carcinoma samples, respectively, and their presence correlated with reduced overall survival in univariate analysis (ezrin, p=0.0189; moesin, p=0.0351). In multivariate analysis (including FIGO stage, residual tumor, histological type, and Silverberg grading), ezrin still remained significant as an independent risk factor (relative risk, 2.39; p=0.012). In SKOV-3 cells, siRNA against ezrin but not moesin inhibited in vitro invasion. These data imply that ezrin is necessary for tumor cell invasion, and the better prognosis of ovarian carcinomas lacking ezrin is probably related to their impaired invasion.

Cell Line, Tumor↗

PubMed2005

Ets1 is an effector of protein kinase Calpha in cancer cells.

PKCalpha and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCalpha expression (siPalpha) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPalpha interferes with both Ets1 protein synthesis and stability. The effect of siPalpha on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCalpha-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCalpha/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCalpha- and Ets1-specific siRNAs (siE1). While only siPalpha induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPalpha and siE1. The data suggest that Ets1 serves as an effector for PKCalpha to fulfil certain functions in cancer cells.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗

PubMed2005

A polymorphism at codon 133 of the tumor suppressor RASSF1A is associated with tumorous alteration of the breast.

The tumor suppressor gene RASSF1A is inactivated or mutated in different tumor entities including breast cancer. The frequency of the genomic variants of RASSF1A in patients with breast tumors has not been evaluated. We studied the association between ten nucleotide polymorphisms of RASSF1A and the risk of breast cancer in 178 cases with tumorous alterations of mammary tissue (including 141 carcinomas and 37 fibroadenomas) and 70 controls by SSCP and sequencing. Polymorphisms of RASSF1A were found at codon 28 and codon 133. The distribution of polymorphisms at codon 28 showed no significant difference between the patient groups: 5 of 178 (2.8%) in patients with tumorous alterations and 2 of 70 (2.9%) in control patients. However, the Gright curved arrow T polymorphism (GCTright curved arrow TCT; Alaright curved arrow Ser) at codon 133, which alters the microtubule association and stabilization domain of RASSF1A, exhibited a different genotype distribution: 29 out of 141 (20.6%) patients with breast carcinoma and 9 out of 37 (24.3%) patients with fibroadenoma harbored mutant T-alleles. However, only in 2 out of 70 (2.9%) controls, the mutant T-allele was detected and therefore the frequency was significantly diminished compared to tumorous alterations (Fisher's exact test: carcinomas vs. controls, p = 0.0003; fibroadenoma vs. controls, p = 0.001). From five probands with homozygous TT-genotype at codon 133, three were diagnosed with carcinomas and two with fibroadenomas. Our data indicate that the mutant T-allele of RASSF1A at codon 133 is correlated with an increased number of breast tumors.

Adult↗

PubMed2004

A conserved sequence in the mouse variable T cell receptor alpha recombination signal sequence 23-bp spacer can affect recombination.

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V-gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23-bp spacer. Using gel-shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre-B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG-binding activity, three proteins were identified: G3BP1 (a nucleic-acid-binding protein with a proposed helicase activity) and two proteins from the high-mobility group (HMG) family--HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR alpha delta locus.

Animals↗

PubMed2003

The biology of the Ets1 proto-oncogene.

The Ets1 proto-oncoprotein is a member of the Ets family of transcription factors that share a unique DNA binding domain, the Ets domain. The DNA binding activity of Ets1 is controlled by kinases and transcription factors. Some transcription factors, such as AML-1, regulate Ets1 by targeting its autoinhibitory module. Others, such as Pax-5, alter Ets1 DNA binding properties. Ets1 harbors two phosphorylation sites, threonine-38 and an array of serines within the exon VII domain. Phosphorylation of threonine-38 by ERK1/2 activates Ets1, whereas phosphorylation of the exon VII domain by CaMKII or MLCK inhibits Ets1 DNA binding activity. Ets1 is expressed by numerous cell types. In haemotopoietic cells, it contributes to the regulation of cellular differentiation. In a variety of other cells, including endothelial cells, vascular smooth muscle cells and epithelial cancer cells, Ets1 promotes invasive behavior. Regulation of MMP1, MMP3, MMP9 and uPA as well as of VEGF and VEGF receptor gene expression has been ascribed to Ets1. In tumors, Ets1 expression is indicative of poorer prognosis.

Animals↗

PubMed2003

Ets-1 activates parathyroid hormone-related protein gene expression in tumorigenic breast epithelial cells.

Parathyroid hormone-related protein (PTHrP) is produced by many tumors not associated with humoral hypercalcemia, including breast cancers. In this study, we used three human immortalized mammary epithelial cell lines that differ in tumorigenicity and PTHrP expression. Using RT-PCR we investigated 5' and 3' alternative splicing of PTHrP transcripts and promoter usage in the lines. Increased levels of P3-derived transcripts and the 1-139 mRNA isoform were observed in the most tumorigenic cell line. Transient transfection experiments identified elements close to P3 promoter that appeared to account for a portion of differential PTHrP expression among the three cell lines. Using site-directed mutagenesis, a previously described Ets-1/Sp1 binding site upstream of P3 was determined to be crucial for full activity of this promoter. RT-PCR and western blot evaluation of Ets family member expression found that Ese-1 was present in all three lines, but that appreciable levels of Ets-1 protein were present exclusively in the most tumorigenic line. Cotransfection of Ets-1 expression vectors activated PTHrP reporter constructs in the most tumorigenic line but not in the other cell lines. These findings suggest a potential mechanism by which PTHrP transcription may be regulated as a consequence of events that promote tumorigenic behavior in breast epithelial cells.

Alternative Splicing↗

PubMed2003

Ets2 and protein kinase C epsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells.

Parathyroid hormone-related protein (PTHrP) promotes the metastatic potential and proliferation of breast cancer cells, and acts anti-apoptotically. In invasive MDA-MB-231 breast cancer cells, transforming growth factor beta-regulated PTHrP synthesis is mediated by an Ets1/Smad3-dependent activation of the PTHrP P3 promoter. In the present study, we studied the regulation of PTHrP expression in non-invasive, Ets1-deficient and transforming growth factor beta-resistant MCF-7 cells. We found PMA to be a strong stimulator of P3-dependent PTHrP expression in MCF-7 cells. Mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase 1 (MEK-1)/ERK1/2 inhibitor PD98059 interfered with this activity. Promoter studies revealed that the PMA effect depended on the Ets and stimulating protein-1 (Sp1)-binding sites. Of several Ets factors tested, Ets2, but not Ese-1, Elf-1 or Ets1, supported the PMA-dependent increase in promoter activity. PD98059 and a threonine to alanine mutation of the ERK1/2-responsive Ets2 phosphorylation site at position 72 inhibited the Ets2/PMA effect. Activated protein kinase C (PKC) epsilon could mimic PMA by stimulating the P3 promoter alone or in co-operation with Ets2 in an MEK-1/ERK1/2-dependent manner. Activated PKC alpha, although capable of co-operating with Ets2, failed to induce transcription from the P3 promoter on its own. The Ets2/PKalpha synergistic effect was neither sensitive to PD98059 nor to Thr(72)/Ala(72) mutation. PMA neither increased the expression of Sp1 nor modulated the transcriptional activity of Sp1. However, it induced the displacement of a yet unknown factor from the Sp1-binding site, which may result in Sp1 recruitment to the promoter. Our results suggest an ERK1/2-dependent Ets2/PKC epsilon synergism to be involved in PTHrP expression in MCF-7 breast cancer cells.

Base Sequence↗

PubMed2003

Interfering with TGFbeta-induced Smad3 nuclear accumulation differentially affects TGFbeta-dependent gene expression.

BACKGROUND: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase. RESULTS: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors. CONCLUSION: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.

Active Transport, Cell Nucleus↗

PubMed2003

Protein kinase Calpha regulates Ets1 transcriptional activity in invasive breast cancer cells.

We have previously shown that PKC inhibitors interfere with the Ets1/Smad3-dependent regulation of parathyroid hormone-related protein (PTHrP) P3 promoter activity by TGFbeta in invasive MDA-MB-231 breast cancer cells. By examining PKC expression in a variety of breast cancer cell lines, the protein level of PKCalpha was found to be much higher in Ets1-expressing MDA-MB-231 and MDA-MB-435 breast cancer cells than in Ets1-deficient MCF-7 and SK-BR3 cells. No correlation of Ets1 expression with the expression of other PKC subtypes (PKCbeta1, PKCbeta2, PKCdelta or PKCepsilon) could be observed. In contrast to MDA-MB-231 cells, PKCalpha-deficient MCF-7 cells do not support Ets1-induced activation of the PTHrP P3 promoter suggesting that PKCalpha may be important for Ets1 activity. A constitutively active form of PKCalpha was found to potentiate the P3 promoter activation by Ets1 alone and in synergy with Smad3. PKCalpha, but not PKCepsilon, also induced phosphorylation of the Ets1 protein. Both PKCalpha effects on Ets1 depended on the exon VII domain of Ets1. Using verapamil and ionomycin, we could show that PKCalpha induces Ets1 phosphorylation independent of calcium mobilization. Collectively, our data suggest that PKCalpha may regulate Ets1 activity in invasive breast cancer cells.

Blotting, Western↗

PubMed2003

Splicing variant DeltaVII-Ets1 is downregulated in invasive Ets1-expressing breast cancer cells.

Transcription factor Ets1 is expressed in invasive breast cancer cells. In T-cells, the splicing variant DeltaVII-Ets1 is naturally produced along with full length Ets1 (fl-Ets1). Though its function is unknown, the lack of important inhibitory domains predicts a regulatory role of DeltaVII-Ets1 in fl-Ets1-controlled transcription. Examining the expression status of DeltaVII-Ets1 in invasive Ets1-producing MDA-MB-231 breast cancer cells, we found that the DeltaVII-Ets1 protein could only be detected when nuclear proteins from these cells were fractionated by ionic exchange chromatography. When overexpressed, DeltaVII-Ets1 was found to be partially degraded in breast cancer cells, but not in Jurkat T-cells or SK-Mel melanoma cells. In contrast, no proteolytic products resulted from ectopic expression of fl-Ets1 suggesting that breast cancer cells are able to specifically cleave DeltaVII-Ets1. Overexpression of DeltaVII-Ets1 reduced survival of MDA-MB-231 cells, but not of MCF-7 cells. A mutant version of DeltaVII-Ets1, lacking first 129 N-terminal amino acids, had no effect. These data suggest that Ets1-producing invasive breast cancer cells specifically downregulate DeltaVII-Ets1, as it may be able to adversely affect the survival of these cells.

Alternative Splicing↗

PubMedDate not supplied

Prognostic and predictive impact of soluble epidermal growth factor receptor (sEGFR) protein in the serum of patients treated with chemotherapy for metastatic breast cancer.

BACKGROUND: A soluble fragment of the epidermal growth factor receptor (EGFR) extracellular domain (sEGFR) can be detected in the serum of cancer patients, but the role of sEGFR is still unclear. MATERIALS AND METHODS: Blood samples from patients receiving chemotherapy for metastatic breast cancer were collected before (n = 101) and after 3 courses of therapy (n=39). Levels of sEGFR and serum HER-2/neu extracellular domain (ECD) were determined by standardized ELISA. RESULTS: A higher percentage of cancer patients (15%) showed sEGFR values below 45ng/mL compared with control subjects (3%, p<0.001). Patients with sEGFR levels below 45 ng/mL showed a trend towards shorter overall survival (median 11.7 versus 15.4 months, p=0.08), which was more pronounced in patients with estrogen receptor-positive primary tumors (median 9.6 versus 15.4 months, p=0.022) Patients with low sEGFR and elevated serum HER-2/neu ECD (>15 ng/mL) also showed a shorter overall survival than those with normal values for both parameters (7.1 versus 15.4 months, p=0.03). Again, this difference was higher in patients with estrogen receptor-positive tumors (4.6 versus 15.4 month, p<0.0001). During treatment, a decrease of sEGFR levels occurred in 74.4% of the patients (p=0.014). CONCLUSION: Low sEGFR levels in patients with metastatic breast cancer are associated with a shorter overall survival, particularly in patients with estrogen receptor-positive tumors. Chemotherapy frequently induces a decrease of sEGFR. The combined, determination of sEGFR and serum HER-2/neu ECD also delivers relevant information. These findings suggest that the sEGFR status in metastatic breast cancer could be of clinical relevance.

Antineoplastic Combined Chemotherapy Protocols↗