Rainald Knecht

Downregulation of human polo-like kinase activity by antisense oligonucleotides induces growth inhibition in cancer cells

A central role for polo-like kinases (PLK) in regulating several stages of mitotic progression has been born out in several species. Overexpression of PLK1 is observed in the majority of hitherto analysed human tumors. PLK1 overexpression is a negative prognostic factor in patients suffering from non-small cell lung cancer, head and neck tumors, esophageal carcinomas and melanomas. In order to define the role of PLK1 for mitotic progression of human cells and for neoplastic cell growth, phosphorothioate antisense oligonucleotides (ASOs) were tested to selectively downregulate PLK1 expression in MDA-MB-435 (breast cancer), HeLa S3 (cervical carcinoma) and A549 (non-small cell lung cancer) cells. ASOs were identified which suppress PLK1 mRNA and protein in a dose-dependent and sequence-specific manner. This approach also led to reduced PLK1 serine/threonine kinase activity. Downregulation of cellular PLK1 levels in cancer cells altered cell cycle progression moderately with an elevated percentage (20–30%) of cells in G2/M. Furthermore, cells with reduced PLK1 protein gained a rounded phenotype with multiple centrosomes. Moreover, ASO treatment resulted in potent antiproliferative effects in cell culture. Considerable antitumor activity was observed in vivo against A549 cells. This study suggests that antisense inhibitors targeted against PLK1 at well tolerated doses may be considered as a cancer therapeutic agent.

Quantitative measurements of perfusion and permeability of oropharyngeal and oral cavity cancer, recurrent disease, and associated lymph nodes using first-pass contrast-enhanced computed tomography studies.

Objectives:We sought to evaluate the routine clinical use of perfusion computed tomography in the detection and differentiation of primary and recurrent oropharynx and oral cavity tumors as well as of nodal disease. Materials and Methods:A total of 77 patients with primary cancer as well as suspected recurrent disease and lymph nodes were evaluated. A dynamic acquisition (4 × 6-mm slices) of the largest axial tumor surface was performed and the tumor blood flow (BF), blood volume (BV), and mean transit time (MTT) were calculated by using a modified deconvolution-based analysis taking into account the extravasation of the contrast agent for permeability surface area product imaging (PS). Tumor volume was calculated and region of interest analysis was performed on the pathologic and normal tissue. Results:The mean BF, BV, and PS values in the primary tumors (77.48 mL/min/100 g tissue; 5.29 mL/min; 13.33 mL/min/100 g tissue, respectively) were highly significantly different (P < 0.01) than those obtained in the normal structures. Mean MTT values (9.01 seconds) also were significantly lowered in the tumors compared with normal tissue (P < 0.05). There was no statistical difference in the perfusion values between the primary and the recurrent tumors. Recurrent disease could be differentiated on the basis of BF (P < 0.05) from tissue changes after chemo-radiation-treatment (mean BF: 69.71 versus 45.31 mL/min/100 g tissue, respectively). Differentiation of the lymph nodes was not possible by means of perfusion values. Tumor volume did not significantly correlate with any perfusion parameter. Conclusions:Perfusion CT of oropharyngeal and oral cavity cancer in clinical routine is feasible and helps outlining the malignant tissue as well as differentiating recurrent disease from nonspecific post-therapeutic changes.

PLK (polo-like kinase), a new prognostic marker for oropharyngeal carcinomas.

The worldwide annual incidence of squamous cell carcinomas of the head and neck (HNSCCs) is about 500,000. The prognosis of these patients is based mainly on the clinicopathological tumor stage, in particular the lymph node status pN, even though it is generally accepted that outcomes can be different among tumors of the same stage. Therefore, our study focused on the identification of molecular parameters for the improvement of the daily clinical diagnosis, which contributes to further prognostic differentiation. Polo-like kinases (PLKs) have been implicated in the regulation of the eukaryotic cell cycle (Lane and Nigg, 1997). Expression of PLK mRNA, a novel marker for cellular proliferation, correlates with the prognosis of patients suffering from different types of tumor (Holtrich et al.,1994; Wolfet al.,1997; Knechtet al.,1999). We have examined the prognostic role of PLK-protein expression in human cancer. Primary tumors from 157 patients with HNSCC, collected over a period of 1 year, were evaluated for PLK immunoreactivity. Therapeutic and post-therapeutic investigations (5 years) of patients (115 male, 42 female) suffering from oropharyngeal carcinomas (stage I5 14, stage II5 30, stage III5 34, stage IV5 79) were performed as follows. Patients in tumor stages I and II underwent surgery, whereas patients in stages III and IV were subjected to surgery and post-operative radiotherapy (2 Gy fractions given as once-a-day treatment to a total dose of 70 Gy applied to the primary region and the neck). Chemotherapy was given non-concomitantly, using 3 cycles of cisplatin (20 mg/qm) and 5-fluorouracil (1,000 mg/qm) according to a standard schedule (Forastiere t al., 1992). Tumor recurrences were treated chemotherapeutically with the same regimen until no response or progressive disease was measured in 2 dimensions by computed tomography. The length of follow-up was between 5 and 132 months. Paraffin-embedded tumor sections were immunolabeled with an affinity-purified PLK-specific, polyclonal rabbit serum. The primary antibody, diluted 1:80, was visualized by the APAAP technique; sections were counterstained with hematoxylin. For comparative purposes, sections were labeled with antibodies for Ki67 (1:50; Dako, Hamburg, Germany) and PCNA (1: 1,000; Dako) using the same technique. Three independent investigators calculated the number of tumor cells (stained nuclei) positive for PLK, Ki67 or PCNA, respectively, per 5,000 tumor cells. A minimum of 15 fields (400 3) per tumor were investigated. Prognostic evaluations were based on the mean PLK index (positive cells/5,000 cells 3 100). Levels of PLK protein in microscopically normal oropharyngeal mucosa (median 5 12.0%, minimum5 0.1%, maximum5 41.0%) surrounding a tumor were low. In 10% of the investigated cases, we observed in the surrounding tissue (within 3 to 5 cm of the tumor) dysplasia (grade I/II). Under these conditions, levels of PLK-protein expression were slightly elevated. Compared to the periphery, PLK protein was over-expressed (median 5 59.4%, minimum5 22.3%, maximum5 74.8%) in oropharyngeal carcinomas (Wilcoxon’s test, p 5 0.016). A correlation of PLK expression with clinicopathological parameters led to further significant correlations with pathological tumor stage ( p 5 0.007) and pN stage ( p 5 0.012) but not with N stage ( p 5 0.09, Kruskal-Wallis test). A KaplanMeier analysis based on a median cut-off showed that patients exhibiting moderate PLK expression ( ,59.4%) had longer survival times than those exhibiting high expression (

Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo.

59.4%, log rank p 5 0.013; Fig. 1). Median survival times were 52 months [95% confidence interval (CI) 36–67 months] and 26 months (95% CI 14–37 months), respectively. To test the selectivity of PLK prognostic information, we performed a Cox regression analysis. During stepwise backward selection from entered variables (age, sex, pT, pN, PLK expression, therapy), only pN (eB5 1.87, 95% CI 1.14–2.89, p 5 0.007) and PLK (eB 5 2.31, 95% CI 0.96–5.56, p 5 0.039) were significant predictors of survival. In contrast, the proliferation markers Ki67 and PCNA, which are frequently discussed as prognostic parameters for HNSCC, were not associated with tumor stage, pN or survival in our study, which is in line with the results of Sommer and Olofsson (1997). Based on immunohistochemical methods, PLK appears to be a new prognostic marker for the daily routine diagnosis of the risk of dying for patients with oropharyngeal carcinoma in addition to the pN stage. Moreover, if the pN stage is unknown

Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells

Cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is critical for the initiation of mitosis. Accumulating data indicate that the deregulation of cyclin B1 is tightly linked to neoplastic transformation. To study the phenotype and the potential preclinical relevance, we generated HeLa cell lines stably transfected with the plasmids encompassing short hairpin RNA (shRNA) targeting cyclin B1. We demonstrate that the reduction of cyclin B1 caused inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis. Cells, entering mitosis, were impaired in chromosome condensation and alignment. Importantly, HeLa cells with reduced cyclin B1 were more susceptible to the treatment of small interfering RNA targeting Polo-like kinase 1 (Plk1) and to the administration of the chemotherapeutic agent taxol. Finally, HeLa cells with reduced cyclin B1 showed inhibited tumor growth in nude mice compared to that of control cells. In summary, our data indicate that cyclin B1 is an essential molecule for tumor cell survival and aggressive proliferation, suggesting that the downregulation of cyclin B1, especially in combination with other molecular targets, might become an interesting strategy for antitumor intervention.

Optimized speech understanding with the continuous interleaved sampling speech coding strategy in patients with cochlear implants : Effect of variations in stimulation rate and number of channels

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.

Results of Cochlear Implantation in Patients with Severe to Profound Hearing Loss—Implications for Patient Selection

The purpose of this study was to investigate the effect of systematic variations in stimulation rate and number of channels on speech understanding in 13 patients with cochlear implants who used the continuous interleaved sampling speech coding strategy. Reducing the stimulation rate from 1,515 to 1,730 pulses per second per channel to 600 pulses per second per channel resulted in decreased overall performance; the understanding of monosyllables and consonants was more affected than the understanding of vowels. Reducing the number of active channels below 7 or 8 channels decreased speech understanding; the identification of vowels and monosyllables was most affected. We conclude that vowel recognition with the continuous interleaved sampling strategy relies on spectral cues more than on temporal cues, increasing with the number of active channels, whereas consonant recognition is more dependent on temporal cues and stimulation rate.

Patient-based cross-platform comparison of oligonucleotide microarray expression profiles

In patients with some residual hearing and minor benefit from conventional hearing aids, the benefits of cochlear implantation have to be weighed carefully against eventual adverse effects. In this study, pre- and post-operative thresholds as well as functional results after cochlear implantation are reported; 17 of 44 implanted adults had residual hearing pre-operatively (mean threshold(250 to 4000 Hz): 106 dB HL) in the implanted ear. Residual hearing in the implanted ear could not, in general, be preserved post-operatively. Seventeen of 44 implanted children had some amount of residual hearing in the implanted ear pre-operatively (implanted ear: 114 dB HL; contralateral ear: 109.9 dB HL; mean thresholds(250 to 4000 Hz))). Contrary to the results in adults, residual hearing in the implanted ear remained statistically unchanged. Hearing in the contralateral ear increased significantly from 109.9 to 101.9 dB HL post-operatively. This increase was mainly attributed to maturation of the central auditory pathway. In adults with residual hearing, the monosyllable word recognition scores increased significantly from 9 per cent pre-operatively to 42 per cent post-operatively. Children with residual hearing tended to perform better on speech-related test material compared to children without prior auditory experience. Cochlear implantation is indicated in adults and children with residual hearing and minor benefit from conventional amplification. The contralateral ear in children should be considered for additional acoustical stimulation.

Demands on caring relatives of head and neck cancer patients

The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether interplatform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general.

In vivo proton MR spectroscopy of primary tumours, nodal and recurrent disease of the extracranial head and neck

Background: Relatives of cancer patients experience high levels of stress that influence the quality of life of these individuals. To investigate whether there is a necessity for simultaneous supportive care of patient relatives, we performed for the first time a study asking the closest relatives of head and neck cancer patients about their needs during and after the treatment to consider how to optimize the situation for such patient groups.