[From tumor tissue via primary cultures to xenograft models: a functional approach in prostate cancer research]. / Vom Gewebe über die Primärzellkultur zum Xenograftmodell : Patientennahe und funktionelle Prostatakarzinomforschung.

The clinical course of prostate cancer, the most common cancer in men, is very variable. Despite intense research activities over the years and besides histopathological criteria, prognostic markers that reliably predict tumor behavior and the necessity for treatment are still missing. A likely explanation for this fact is the lack of good tumor models, mimicking the in vivo situation. These models are not only essential for a better understanding of the pathogenesis of prostate cancer but also play an important role in the development of new therapeutic strategies. Since results of permanent cell culture experiments reflect only in part real tumor behavior and primary cultures from patient material cannot be grown indefinitely, novel approaches need to be developed to achieve reliable and clinically relevant prostate cancer research.In this work the development of several approaches for culturing primary prostate cancer tissue is illustrated and a forecast of future research plans utilizing xenograft models in mice is made.

[Educational initiative of the German Society of Urology for innovative research : Workshops 2009]. / Ausbildungsinitiative der Deutschen Gesellschaft für Urologie für innovative Forschung : Workshops 2009.

The tasks of the Working Group on Urological Research (AuF) of the German Society of Urology (DGU) are to support communication and initiation of joint ventures in German urology and to cooperate with associated subjects and neighboring countries. The annual "wet lab workshops" needs a space between annual and "wet lab workshops" on the topics of tumor cell culture, gene silencing, proteomics, and tissue engineering and the use instead of annual topic-related symposium"urological research," organized and carried out by the AuF as of 2009, serve to achieve a close change to closer integration of praxis and theory. This should contribute to a lasting quality improvement of the scientific work in urology. Accomplishing these objectives seems urgently necessary to preserve the interests of urologists, because more than ever research has become indispensable in an increasingly difficult environment of health care policy.

[Development of a three-dimensional primary prostate cancer cell culture model]. / Entwicklung eines dreidimensionalen Prostatakarzinomzellkulturmodells.

Much prostate cancer research is based on cell culture results. Recent genomic studies found major differences between primary prostate cancer tissue and established prostate cancer cell lines, which calls into question the clinical relevance of study results based on cell cultures.Using primary cultures of prostate cancer cells from prostatectomy specimens seems to be a reasonable solution, but primary cell cultures are much more difficult to establish. In this study, a primary cell culture model was combined with an invasion assay. With this combination it was possible not only to select invasive cell clones from the primary culture but also to culture these cells in a three-dimensional model, forming spheroids. A further characterization of this cell population was done by comparative genomic hybridization, showing numerous genetic alterations. The presented cell culture model offers, for the first time, an opportunity to isolate invasive growing cells from primary prostate cancer tissue and cultivate these cells for further analyses.

An improved in vitro model to characterize invasive growing cancer cells simultaneously by function and genetic aberrations.

Invasion into the surrounding tissue and bone metastasis is a common feature of advanced prostate cancer. Chromosomal and other genetic or epigenetic abnormalities were aligned to this behaviour mostly by using permanent cell lines, paraffin embedded tissue or primary tumour samples. Both attempts fail to reflect either the original situation or functional information in the patient's tissue. Thus, we developed an improved in vitro assay to follow invasion of prostate cancer cells derived from fresh samples of radical prostatectomy specimens. Fresh tumour samples were applied onto Matrigeltrade mark-coated invasion chambers using a cocultivation model. Invasive growing cells were harvested from the bottom of the membrane or from the underlying gel and further characterized using comparative genomic hybridization. Prostate cancer cells have the capability to invasively grow through the barrier of a Matrigeltrade mark and could easily be sampled in a pad of Matrigeltrade mark. Comparative genomic hybridization revealed characteristic chromosomal aberrations of the invasive growing cells. Noteworthy is their ability to spheroid formation, which allows for further cell propagation by standard cell culture methods. Thus, our improved invasion model is a tool for the sampling of invasive growing cancer cells from fresh human tumour material allowing for functional as well as genetic studies.

[Proteasome inhibitors: induction of apoptosis as new therapeutic option in prostate cancer]. / Proteasomeninhibitoren: Apoptoseinduktion als neue Therapieoption beim Prostatakarzinom?

New perspectives in prostate cancer genesis and putative clinical management have emerged in recent years . Apoptosis plays a major role in this environment. Proteasome inhibitors block the action of a multicatalytic proteinase complex involved in the degradation of intracellular proteins, particularly with regard to cell cycle regulation and apoptosis. Numerous in vitro studies have demonstrated the ability of these compounds to induce apoptosis and enhance the activity of conventional tumoricidal agents in many cancer cell types, including prostate cancer cells. They point out the use of these potent inhibitors as a new potential molecular approach to the therapeutic management of prostate cancer. Furthermore, the action of proteasome inhibitors has been tested in animal models and in patients with hormone refractory prostate cancer, resulting in both PSA and tumor volume decrease. PS-341 (bortezomib, Velcade) is the first proteasome inhibitor with clinical application in cancer therapy that has been used in clinical trials to date. This report reviews the current status of those papers that have tried to analyze the connection between the proteasome pathway and apoptosis. We present our results of proteasome inhibition in individual prostate cancer cell lines. Proteasomal inhibition may offer a new therapeutic access in "molecular targeting" of prostate cancer.

Proteasome inhibitors and their combination with antiandrogens: effects on apoptosis, cellular proliferation and viability of prostatic adenocarcinoma cell cultures.

The 26S proteasome is a ubiquitin-dependent proteolytic system that has been implicated in the regulation of cell cycle progression and apoptosis. We investigated the effects of the proteasome inhibitors MG115 and PSI alone or in combination with different concentrations of the antiandrogen hydroxyflutamide on the cellular proliferation, apoptosis and viability of 10 prostatic adenocarcinoma cell cultures. Treatment with both proteasome inhibitors resulted in apoptosis induction, whereas the combinations with hydroxyflutamide generally did not, with the exception of MG115 combined with 10(-7) M hydroxyflutamide. MG115 caused a significant decrease in cellular proliferation, as did the combinations of both proteasome inhibitors with hydroxyflutamide, whereas hydroxyflutamide alone was only effective at a concentration of 10(-5) M. Cellular viability was significantly reduced when both proteasome inhibitors were combined with 10(-5) M hydroxyflutamide. Although the results varied among different cell lines, we conclude that proteasome inhibitors are able to induce apoptosis and reduce cellular proliferation. They might prove effective as antineoplastic substances in prostatic adenocarcinoma alone or in combination with antiandrogens.

Cell adhesion molecule CEACAM1 associates with paxillin in granulocytes and epithelial and endothelial cells.

CEACAM1 functions as an epithelial tumor suppressor and as an angiogenic growth factor. In the present study, utilizing differentially (serine/threonine or tyrosine) phosphorylated cytoplasmic domains of CEACAM1 and CEACAM3 as bait to isolate associated proteins from granulocyte extracts, we have identified human paxillin as a binding partner of the tyrosine-phosphorylated cytoplasmic CEACAM1 domain. CEACAM1-paxillin complexes were coimmunoprecipitated from extracts of granulocytes, the colonic cell line HT29, and HUVECs. We identified phosphorylated Tyr-488-a residue in the cytoplasmic CEACAM1 domain known to be essential for the tumor suppressive effect-to be necessary for this association. The CEACAM1-paxillin interaction was confirmed using laser scanning confocal microscopy analyses in granulocytes and HT29 cells, where CEACAM1 colocalizes with paxillin at the plasma membrane. In HUVECs a highly polarized expression pattern and colocalization of paxillin and CEACAM1 was observed. These findings support the findings that CEACAM1 is linked to the actin-based cytoskeleton.

Expression of the human telomerase reverse transcriptase is not related to telomerase activity in normal and malignant renal tissue.

In this study, the association between telomerase activity and the expression of the human telomerase subunits human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in paired neoplastic and normal renal tissue samples was investigated. Reverse transcription (RT)-PCR on 20 tumor nephrectomy samples revealed that hTR was constitutively expressed both in cancer and normal tissue samples, independent of the telomerase activity status. Remarkably, using in situ hybridization, the expression levels of hTR were found to be markedly higher in the normal tissue than those in the tumors. Expression of hTERT mRNA by RT-PCR was observed in 90% of the cancer samples and, notably, also in 75% of the corresponding normal renal tissue samples. Because all of the normal tissue samples and some of the tumor samples were shown to be telomerase negative, our findings suggest that hTERT mRNA expression is not sufficient for telomerase enzyme activation. Furthermore, semiquantitative RT-PCR revealed equal or even higher hTERT mRNA expression levels in the telomerase-negative normal samples than in the corresponding cancer samples with telomerase activity, contradicting the assumption that a certain threshold level of hTERT mRNA is required for telomerase activation at least in renal tissue. It seems more likely, that other mechanisms, such as posttranscriptional modification of hTERT or inactivation of telomerase inhibitors, are involved in the acquisition of enzyme activity.

Two different forms of p53 localized differently within cells of urogenital tumours.

We analyzed the subcellular localization of p53 in prostate and bladder carcinoma cells. Using laser scanning microscopy and PAb1620, a monoclonal antibody recognizing the wildtype conformation of p53, and another monoclonal antibody directed against the mutant conformation of the protein (PAb240), we found two different subsets of p53 within the same cell. The wildtype subgroup was found in the nucleolus, whereas the mutant protein was confined to the nucleus. The results obtained by immunofluorescence were verified by Western blot analysis and immunoprecipitation. Thus, our findings demonstrate an unusual subcellular localization pattern of p53 in prostate and bladder cancer cells which may indicate another mechanism of inactivation of p53.

p53 autoantibodies in patients with urological tumours.

OBJECTIVE: To determine the presence of p53 serum antibodies in patients with clinically well-defined urological cancer using a new enzyme-linked immunosorbent assay (ELISA). PATIENTS AND METHODS: The study included 73 patients with prostatic cancer, 72 with transitional cell carcinoma of the urinary tract, 37 with renal cell cancer and 16 controls with a benign disease, all of whom were tested using the ELISA for p53 autoantibodies. The specific reaction of the ELISA (positive p53 antibody titre) was confirmed by Western Blot analysis. RESULTS: Thirteen patients with cancer and one control patient (7.6% overall) were positive for p53 autoantibodies. The sensitivity of the test was low, whereas the specificity was remarkably high. Surprisingly, 9 of the 13 p53-positive patients died within a median of 3.7 months (range 2-6) and the one positive control patient died of undetected lung cancer. There was no significant correlation of p53 antibody positivity with clinical stage or tumour-specific differences. CONCLUSIONS: The expression of p53 autoantibody seems to be a very late but significant event in urological tumour development, with the worst outcome (tumour-specific death) within a few weeks of developing positivity. In histopathologically heterogeneous tumour entities, p53 autoantibodies might be independent prognostic factors in patients with urological cancers.

A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography.

Confocal microscopy and vital fluorescence techniques were combined for the first time to investigate ex vivo human dental plaque. The vital fluorescence technique used discriminates vital from dead cells, while confocal laser scanning microscopy allows the optical sectioning of undisturbed biofilms leaving the samples intact during analysis. The concomitant use of both methods made an examination of the three-dimensional architecture of dental plaque possible. The topography of plaque biofilms that were allowed to accumulate in situ on glass and enamel was recorded. The distribution of plaque microflora vitality as well as its accumulation varied according to plaque age. A plaque thickness of up to 8, 35 and 45 microm was estimated ex vivo on enamel after 1, 2 and 3 days, respectively. Young and sparse plaque biofilms consisted mainly of dead material. Vital bacteria were observed on top of this dead layers.

Androgen receptor expression, proliferation index and aneuploidy in tissue explant cultures derived prostate carcinoma cells co-cultivated on membranes.

OBJECTIVES: An improved explant cell culture technique to avoid selection of prostatic adenocarcinoma cells toward diploid cells is described. MATERIAL AND METHODS: 21 prostatic carcinoma specimens which were obtained from 13 primary prostatic adenocarcinomas after radical prostatectomy were cultivated. Ploidy of the cells was monitored by fluorescence in situ DNA hybridization using the centromere-specific DNA probes pUC1.77, p alpha 7t1 and pY3.4. Phenotypic examination of androgen receptor (AR) expression was performed simultaneously with immunostaining by Ki-67 as proliferation marker to identify androgen-independent growing cell clones. RESULTS: Interestingly, a high aneuploidy rate of the cell cultures was found with maintenance of aneuploidy in 18 (86%) of the 21 paraffin-embedded cancer tissue specimens with proved aneuploidy. Significant aneuploid cell populations were retained up to a maximum of ten transfer steps. During serial transfer of tumor pieces the aneusomic fraction slightly decreased as well as the percentage of AR/Ki-67-positive cells. CONCLUSIONS: The presented in vitro model allows to study the proliferation of genetically abnormal cells with respect to hormone dependency in a paracrine situation.

A new serial transfer explant cell culture system for human prostatic cancer tissues preventing selection toward diploid cells.

An improved explant cell culture technique to avoid selection of prostatic adenocarcinoma cells toward diploid cells is described. This method is based on 1) histologically characterized tissue explants, 2) the use of polyethylenteraphthalate (PET) membranes as growth surface, which are part of special inserts in six-well-plates to allow 3) cocultivation with heterologous fibroblasts, and 4) coating of the membranes with elements of the extracellular matrix. The main characteristic of this particular approach is the serial transfer of the tissue explant from one membrane to the other. Up to ten serial transfer steps could be performed to produce cell monolayers growing out of the same tissue specimen. Using this approach, 21 prostatic carcinoma specimens that were obtained from 13 primary prostatic adenocarcinomas after radical prostatectomy were cultivated. Ploidy of the cells was monitored by fluorescence in situ DNA hybridization using the centromere specific DNA probes pUC1.77, p alpha 7t1, and pY3.4. Interestingly, a high aneuploidy rate of the cell cultures was found with maintainance of aneuploidy in 18 (86%) of the 21 paraffin-embedded cancer tissue specimens with proved aneuploidy. Although a slight decrease of the proportion of aneuploid cells during serial transfer was observed, significant aneuploid cell populations were retained up to a maximum of ten transfer steps. These findings indicate that selection toward diploid cells can be prevented by improved cell culture techniques that mimic the in vivo situation.

Local production of interleukin-4 during radiation-induced pneumonitis and pulmonary fibrosis in rats: macrophages as a prominent source of interleukin-4.

Fibrosis of lung tissue is a frequent and serious consequence of radiotherapy of mammary carcinoma. The pathogenesis of radiation-induced pulmonary fibrosis remains unclear. Cytokines such as transforming growth factor beta (TGFbeta) and interleukin-4 (IL-4) have been reported to stimulate collagen synthesis in fibroblasts in vitro. The aim of this study was to document the presence of IL-4 during the development of post-irradiation lung fibrosis. Right lungs of male Fischer rats were irradiated with a single dose of 20 Gy and IL-4 expression in the irradiated lungs was monitored for a period of three months. IL-4 gene transcription as determined by ribonuclease protection assay (RPA) as well as IL-4 synthesis as shown by Western blotting increased in the irradiated lungs reaching a plateau concentration within 3 weeks after irradiation. Enhanced IL-4 production was still detected at day 84 after irradiation. The cellular origin of IL-4 was analyzed by in situ hybridization and two-color immunofluorescence on lung tissue sections and on cytospin preparations of leukocytes obtained from bronchoalveolar lavages. These experiments revealed a substantial IL-4 production by macrophages during development of post-irradiation lung fibrosis.

Selection toward diploid cells in prostatic carcinoma derived cell cultures.

For elucidation of the growth-regulatory mechanisms in prostatic carcinoma, in vitro investigations on prostatic cell cultures are required. However, one major problem of cell culturing is the selection of particular cell types such that the cell lines representing only some of the features as compared with the tumor of origin. We studied the chromosomal composition of 20 prostatic tissue-derived cell cultures and 12 original (fresh) tissue specimens that were obtained from 13 patients with prostatic adenocarcinoma. Using fluorescence in situ DNA hybridization (FISH), evident clonal abnormalities were detected in 78% of the fresh cancer samples and in 47% of the cultured cancer samples. Of the seven cases revealing clonal abnormalities in the fresh cancer specimen, aneuploidy was detected in only two samples after cell culturing at the earliest passage studied. The aneuploid cell populations in the cultured samples were all lost during progressive subcultivation (after passage 4). Interestingly, by performing FISH on cytogenetic preparations aneuploidy was confined to the interphases, with the metaphases being found to be diploid. This finding indicates that the aneuploid cells have a proliferation disadvantage in cell culture resulting in an overgrowth of diploid cells.

Phenotypic characterization of androgen receptor-related proliferation in prostatic-carcinoma derived cell-cultures.

Our study reviews the central role of androgens for the development of androgen-insensitive growth in prostatic carcinoma using an individual dynamic model which emulates aspects of proliferation control. Our aim is the identification of individually proliferating cells from prostate carcinoma. Short-term cultured cells (n=205) from radical prostatectomy specimens have been used as the major method to skew the correspondence between the androgen-receptor (AR), proliferation-markers (Ki-67, PCNA) and morphological landmarks (nucleoli, AgNOR). These techniques allowed the identification of distinct androgen-insensitive, proliferating cell-clusters and in the future will allow the analysis of genotypic changes in identified proliferating cell clones.

Different subnuclear localization of wild-type and mutant p53 in human prostate-cancer cells.

The growth suppressor protein p53 is abnormally expressed in a variety of different human tumor cells. We have analyzed the expression of p53 in cell cultures derived from tissues of radical prostatectomies and in the permanent prostate carcinoma cell line PC-3 using two different p53 specific monoclonal antibodies. With the wild-type specific monoclonal antibody PAb1620 we found p53 localized in nucleoli whereas only a few cells were positively stained in the nucleus with the mutant specific monoclonal antibody PAb240. Control experiments with p53 from SV80 cells which express wild-type p53 and HT29 cells expressing mutant p53 documented the specificity of the monoclonal antibodies. The specificity of the antibodies in recognizing indeed p53 was demonstrated further by immunoprecipitation analysis of p53 from the same cell cultures. Since p53 is usually localized to the nucleus our results may represent a specific feature of the wild-type phenotype of p53 in prostate carcinoma cells. The localization of p53 in nucleoli may be another mechanism of the inactivation of wild-type p53.

DNA aneuploidy in prostatic adenocarcinoma: a frequent event as shown by fluorescence in situ DNA hybridization.

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.