Biomedical subjects
Publications and source records attributed to Klaus Strebhardt.
The remarkable progress in the discovery of small molecules that target protein kinases continues, and two recent reports have described alternative approaches to this task: homology modeling and phenotype-based screening. Compounds targeting the nucleotide binding pockets of polo-like kinases in particular provide significant new insights into the molecular mechanisms controlling cell-division phenotypes.
Deregulated cell cycle control is a hallmark of cancer cells. Developmental or other mitogenic stimuli activate the proliferation of normal cells in response to the requirements of growing tissues. In contrast, cancer cells liberate from proliferative restrictions exerted by anti-proliferative signals arising from the stroma and by endogenous genetic programs that correlate to the terminal differentiation of cells. The study of cyclin-dependent kinases (Cdks) and polo-like kinases (Plks) as evolutionary conserved regulators of the cell cycle has contributed significantly to our current understanding of the mechanisms that underlie the proliferation of mammalian cells. Given the importance of Plk1 for mitotic progression the temporal expression of Plk1 is crucial and has to be tightly regulated. It is known that steady-state Plk1 mRNA and protein levels are coordinately regulated during cell cycle progression, being low during interphase but high in mitosis. This review will summarize the current knowledge on how cell cycle-dependent transcriptional regulation of the Plk1 gene is achieved. While binding sites for various transcriptional activators are dispersed throughout the entire Plk1 promoter region, the cell cycle-dependent regulation of the Plk1 gene expression seems to be regulated by G1-specific repression rather than by G2/M-specific activation of the Plk1 transcriptional unit. The tumor suppressor gene p53 was identified as a key player in the precise restriction of Plk1 gene expression to the G2/M phase. The activity of p53 is in turn controlled by Plk1 itself indicating the existence of an auto-regulatory mechanism involved in the cell cycle-dependent regulation of the Plk1 gene. Furthermore, transcription factors regulated by Plk1 will also be subject of discussion.
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RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA.
Polo-like kinase 1 (Plk1) is a key regulator of progression through mitosis. Although Plk1 seems to be dispensable for entry into mitosis, its role in spindle formation and exit from mitosis is crucial. Recent evidence suggests that a major role of Plk1 in exit from mitosis is the regulation of inhibitors of the anaphase-promoting complex/cyclosome (APC/C), such as the early mitotic inhibitor 1 (Emi1) and spindle checkpoint proteins. Thus, Plk1 and the APC/C control mitotic regulators by both phosphorylation and targeted ubiquitylation to ensure the fidelity of chromosome separation at the metaphase to anaphase transition. The mechanisms underlying the control of genomic stability by Plk1 are discussed in this review.
Nanoparticles consisting of human serum albumin (HSA) represent a promising strategy for targeted drug delivery to tumour cells. The coupling of the antibody trastuzumab to HSA nanoparticles takes advantage of the capability of HER2-positive cells to incorporate substances binding to HER2. In our present study, we developed nanoparticles based on HSA which were covalently modified on their surface with thiolated trastuzumab. A special focus was on the optimisation of the thiolation procedure of the antibody under the aspect of an effective binding to particle surfaces. Different thiolation conditions were evaluated and the degree of antibody dimerisation was determined. We analysed the thiolated antibody by size exclusion chromatography (SEC) and identified the best thiolation procedure for the preparation of trastuzumab-conjugated nanoparticles. Over a storage period of 6 weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any changes. Biological activity was confirmed under cell culture conditions: antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis. These data provide the basis for the development of stable and biological active carrier systems for directed targeting of tumour cells using trastuzumab-conjugated HSA nanoparticles.
Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.
Human polo-like kinase 1 (PLK1) is essential during mitosis and in the maintenance of genomic stability. PLK1 is overexpressed in human tumours and has prognostic potential in cancer, indicating its involvement in carcinogenesis and its potential as a therapeutic target. The use of different PLK1 inhibitors has increased our knowledge of mitotic regulation and allowed us to assess their ability to suppress tumour growth in vivo. We address the structural features of the kinase domain and the unique polo-box domain of PLK1 that are most suited for drug development and discuss our current understanding of the therapeutic potential of PLK1.
As a model for hormone-dependent breast cancer, we studied the MCF-7 cell line to examine differences in the stimulation of the inducible (i) and endothelial (e) nitric oxide synthase (NOS) and the role of 17beta-estradiol (E(2)). MCF-7 cells were stimulated with (a) E(2) (10(-8)M) and (b) a combination of different cytokines such as interleukin-1 beta (Il-1beta), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (INF-gamma), and lipopolysaccharide (LPS). e-NOS and i-NOS proteins were measured using Western blot analysis. Using the Griess method nitric oxide (NO) was estimated by assessing the stable product nitrite (NO(2-)) in the culture medium, and a direct method, employing EPR spin trapping also was used. Western blot analysis revealed the presence of e-NOS and i-NOS in MCF-7 cells. In Western blot analysis, e-NOS, but not i-NOS, expression could be stimulated by E(2). An increase in NO(2-) was noted after stimulation of MCF-7 using different combinations of cytokines Il-1beta, TNFalpha and INFgamma, and LPS, but not after E(2). In conclusion, e-NOS and i-NOS are weakly expressed in the MCF-7 cell line, but are stimulated differently. The MCF-7 cell may contain both a constitutive NOS and an inducible NOS.
The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.
RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.
Polo-like kinases (Plks) play pivotal roles in the regulation of cell cycle progression. Plk1, the best characterized family member among mammalian Plks, strongly promotes the progression of cells through mitosis. Furthermore, Plk1 is found to be overexpressed in a variety of human tumors and its expression correlates with cellular proliferation and prognosis of tumor patients. Although all Plks share two conserved elements, the N-terminal Ser/Thr kinase domain and a highly homologues C-terminal region termed the polo-box motif, their functions diverge considerably. While Plk1 is inhibited by different checkpoint pathways, Plk2 and Plk3 are activated by the spindle checkpoint or the DNA damage checkpoint. Thus, Plk2 and Plk3 seem to inhibit oncogenic transformation. Deregulation of Plk1 activity contributes to genetic instability, which in turn leads to oncogenic transformation. In contrast, Plk2 and Plk3 are involved in checkpoint-mediated cell cycle arrest to ensure genetic stability, thereby inhibiting the accumulation of genetic defects. In this review, we shall discuss the roles of Plks in oncogenesis and Plk1 as a target for therapeutic intervention against cancer.
Nitric oxide synthases are expressed in breast cancer. To elucidate the clinical role of the inducible NOS (i-NOS) in human breast cancer, 161 primary breast cancer tissues were stained immunohistochemically. Staining patterns for i-NOS were correlated with classical prognostic factors such as lymph node status, age, hormonal receptor status, tumour size and tumour differentiation. With classical prognostic factors such as lymph node status, age, hormonal receptor status, tumour size and tumour differentiation. Patients survival was also analysed. Sixty-one percent of the tumours stained positively for i-NOS. Detection of i-NOS was positively correlated with increasing tumour size and decreasing tumour differentiation (P=0.018 and P=0.039, respectively). However, in the 50 year age group, i-NOS staining also correlated with lymph node status. Patients with i-NOS-positive breast carcinomas had a significantly worse overall survival rate versus those with negative stains (5-year survival rate 84.8% versus 67.1%; P=0.049; log-rank test). To date, this is the largest analysis of i-NOS expression in breast cancer patients and the only study to assess survival.
RNAi (RNA interference) was originally detected in Caenorhabditis elegans as biological response to exogenous double-stranded RNA (dsRNA), which induces very effective sequence-specific silencing of gene expression. Further investigations revealed that RNAi can occur in many eukaryotic species. Increasing understanding of the biochemical components of RNAi indicates the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer processes the dsRNA to small interfering RNAs (siRNAs) 21-23 nt in length. These siRNAs enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induce destruction of the corresponding mRNAs. Since RNAi has become an excellent strategy for gene silencing, it is tempting to apply this technology to 'knock-down' gene expression in living animals. The generation of transgenic mice from embryonic stem cells expressing small hairpin RNAs (shRNAs) has provided evidence for in vivo application of RNAi. Furthermore, different experimental strategies have been developed to analyze the influence of chemically synthesized siRNAs and of vector-based shRNAs on the expression of different transgenes and endogenous genes in vivo. Recent studies describe the in vivo delivery of siRNAs to inhibit transgene expression in certain organs of adult mice, predominately murine liver. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor-bearing animals with siRNAs are beginning to emerge. They are of utmost interest for systemic diseases such as cancer. In addition, several groups have shown that RNAi can also be used to block the infectivity or suppress the replication of different RNA viruses relevant to human diseases including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). In summary, multiple lines of evidence indicate that RNAi seems to become a powerful tool for the fight against undesirable gene expression in human diseases.
Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.
Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50-80% 48 h after transfection and apoptosis was increased from 5 to 40-50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.
BACKGROUND: RNA interference initiated by small interfering RNAs effectively suppresses gene expression, but the suppression is transient, which limits the therapeutic use of this technique. Polo-like kinase 1 (PLK1) is a key cell cycle regulator that is overexpressed in various human tumors. We used a xenograft mouse model to determine whether an RNA interference-based strategy that used short hairpin RNAs (shRNAs) to suppress PLK1 expression could inhibit tumor growth in vivo. METHODS: HeLa S3 cervical and A549 lung cancer cell lines were transfected with plasmids containing U6 promoter-driven shRNAs against human PLK1 or control (parental or scrambled) plasmids. Plasmids were treated with the nuclease inhibitor aurintricarboxylic acid (ATA) as protection against nucleases in murine blood. Nude mice carrying xenograft tumors were injected with shRNA plasmids, and their xenograft tumor growth was assessed. Northern and western blot analyses were used to measure PLK1 mRNA and protein expression, respectively, in transfected cultured cells and in xenograft tumors. All statistical tests were two-sided. RESULTS: Levels of PLK1 mRNA and protein were lower in HeLa S3 and A549 cancer cells transfected with PLK1 shRNA plasmids than in corresponding cells transfected with control parental or scrambled PLK1S shRNA plasmids. Proliferation of cells transfected with PLK1 shRNA was lower than that of cells transfected with either control plasmid, and proliferation of cells transfected with ATA-treated PLK1 shRNA plasmids was even lower. In mice with human xenograft tumors, PLK1 shRNA expression from ATA-treated plasmids reduced tumor growth to 18% (95% confidence interval [CI] = 12% to 26%; P =.03) and from untreated plasmids reduced tumor growth to 45% (95% CI = 26% to 64%; P =.1) of that of tumors in mice treated with scrambled control PLK1S shRNA plasmids. CONCLUSIONS: The combination of shRNA-mediated gene silencing with effective in vivo gene delivery strategies appears to generate a long-lasting silencing signal.
Nanoparticles consisting of human serum albumin (HSA) and containing different antisense oligonucleotides (ASO) were prepared by desolvation. The preparation process was optimised regarding the amount of desolvating agent, stabilisation conditions as well as nanoparticle purification. The glutaraldehyde crosslinking procedure of the particle matrix was identified as a crucial parameter for biodegradability and drug release of the nanoparticles. The influence of chain length and backbone modification of ASOs on the drug loading efficiency was investigated. The loading increased with longer chain length and employment of a phosphorothioate backbone. The resulting nanoparticles were tested in cell cultures for cytotoxicity and cellular uptake. In different tumour cell lines no cytotoxic effect was observed up to nanoparticle concentrations of 5000 microg/ml. All cell lines showed a significant cellular uptake of HSA nanoparticles. The entrapment of a fluorescent labelled oligonucleotide within the particle matrix was used for the detection of the intracellular drug release of the carrier systems. Confocal laser scanning microscopy revealed that nanoparticles crosslinked with low amounts of glutaraldehyde, rapidly degraded intracellularly, leading to a significant accumulation of the ASO in cytosolic compartments of the tumour cells.