Jürgen Giebel

Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer

Prostate cancer (PCA) is the most common type of cancer found in men of western countries and is the leading cancer death next to lung cancer and colorectal cancer. Prostate-specific antigen (PSA) test is an established diagnostic tool for PCA detection, but confirmation of diagnosis by histopathological evaluation of prostate needle biopsies is performed. To define protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH, n=11) and prostate cancer (PCA, n=12) patients by two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins were identified. The important proteins identified included prostatic acid phosphatase precursor, a significant overexpressed protein in PCA, prohibitin, NDRG1 tumor suppressor proteins, heat shock proteins, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH (n=13), moderate staining in prostate intra-epithelial neoplasia (PIN, n=5) but strong staining in PCA (n=18). Our results demonstrate that protein profiling and mRNA studies can be performed on the same prostate biopsy. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCA and BPH. Some of the interesting proteins identified in this approach may serve to develop new targets for PCA diagnosis and treatment.

Altered expression of tumor protein D52 regulates apoptosis and migration of prostate cancer cells.

Tumor protein D52 (TPD52) is a protein found to be overexpressed in prostate and breast cancer due to gene amplification. However, its physiological function remains under investigation. In the present study, we investigated the response of the LNCaP human prostate carcinoma cell line to deregulation of TPD52 expression. Proteomic analysis of prostate biopsies showed TPD52 overexpression at the protein level, whereas its transcriptional upregulation was demonstrated by real‐time PCR. Transfection of LNCaP cells with a specific small hairpin RNA giving efficient knockdown of TPD52 resulted in significant cell death of the carcinoma LNCaP cells. As demonstrated by activation of caspases (caspase‐3 and ‐9), and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with enhanced green fluorescence protein‐TPD52 or a specific small hairpin RNA. Enhanced green fluorescence protein‐TPD52 overexpressing cells showed an increased proliferation rate, whereas TPD52‐depleted cells showed the reverse effect. Additionally, we demonstrate that exogenous expression of TPD52 promotes cell migration via αvβ3 integrin in prostate cancer cells through activation of the protein kinase B/Akt signaling pathway. From these results, we conclude that TPD52 plays an important role in various molecular events, particularly in the morphological diversification and dissemination of prostate carcinoma cells, and may be a promising target with respect to developing new therapeutic strategies to treat prostate cancer.

Human agmatinase is diminished in the clear cell type of renal cell carcinoma

The proteome of RCC was analyzed by 2D PAGE to search for tumor‐associated proteins. Agmatinase, which hydrolyzes agmatine to putrescine and urea, was identified by mass spectrometry and database searches and shown to be downregulated in tumor cells. Additionally, RT‐PCR and Northern blot analyses demonstrated a clearly decreased amount of agmatinase mRNA in tumor cells. The differential expression of agmatinase mRNA was confirmed at the protein level. Western blot analysis showed almost no detectable agmatinase protein in tumor cells compared to corresponding normal renal tissue. Agmatinase mRNA is most abundant in human liver and kidney but expressed to a lesser extent in several other tissues, including skeletal muscle and small intestine. The human agmatinase gene encodes a 352‐residue protein with a putative mitochondrial targeting sequence at the N‐terminus. Using transfection and immunohistochemical studies, we show that agmatinase is localized in the mitochondria. Immunohistochemical studies revealed that agmatinase in the normal kidney is restricted to tubulus epithelial cells, while in tumors staining was low and heterogeneous. Thus, expression of human agmatinase is altered in RCC. We discuss the consequences of these findings in terms of polyamine, NO metabolism and macrophage function.

Down-regulation of the metastasis suppressor protein KAI1/CD82 correlates with occurrence of metastasis, prognosis and presence of HPV DNA in human penile squamous cell carcinoma

In penile squamous cell carcinoma (PSCC), the outcome largely depends on early detection and resection of inguinal lymph node metastases. We investigated the role of metastasis suppressor protein kang ai 1 (KAI1)/cluster of differentiation 82 (CD82), which is known to be of prognostic significance for a wide variety of cancers. Moreover, we analysed the tumours for human papillomavirus (HPV) DNA and loss of heterozygosity at the 11p11.2 locus. Tissue samples of 30 primary PSCCs were investigated immunohistochemically using an anti-KAI1/CD82 polyclonal antibody. The expression was assessed according to the degree of KAI1/CD82-positive tumour cells as positive, decreased or negative. The presence of HPV6/11, HPV16 and HPV18 DNA was analysed by polymerase chain reaction. All patients with decreased or negative expression of KAI1/CD82 in primary lesions had lymph node metastases (p = 0.0002). Patients with positive KAI1/CD82 expression showed a significant better prognosis for survival compared to the other groups (p = 0.0042). Presence of HPV DNA was associated with decreased or negative KAI1/CD82 expression. Lacking or decreased expression of metastasis suppressor gene KAI1/CD82 appears to be a prognostic parameter for the occurrence of lymph node metastases in PSCC. Our study suggests an association of decreased KAI1/CD82 expression with tumour progression, development of metastases and disease-specific death.

Expression of AP-2α, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression?

Progression of melanoma is associated with loss of the transcription factor AP‐2α and tyrosine‐kinase receptor c‐kit. However, the mechanisms by which these two proteins are down‐regulated have not been fully elucidated. Fifty non‐selected melanomas comprising ten superficial spreading melanomas (five exhibiting a radial growth phase and five a vertical growth phase), ten primary nodular melanomas, 30 melanoma metastases, and 16 naevi were investigated by direct sequencing analysis of the AP‐2α and c‐kit genes and by immunohistochemistry for the respective proteins. Because it has recently been demonstrated that AP‐2α is preferentially cleaved by caspase‐6 and to a lesser extent by caspase‐3, immunohistochemistry for the cleaved (activated) forms of caspase‐6 (c‐casp‐6) and caspase‐3 (c‐casp‐3) was carried out. No mutations were identified in the c‐kit gene, but three different point mutations were demonstrated in the activation motif of AP‐2α in four tumours: one vertical growth phase superficial spreading melanoma, one nodular melanoma, and two metastases. Immunohistochemistry revealed progressive loss of the AP‐2α and c‐kit proteins in primary melanomas and metastases when compared with naevi. The decrease of both markers was more accentuated in the dermal component of all primary tumours, with c‐kit more affected than AP‐2α. All invasive melanomas and metastases expressed c‐casp‐6. c‐casp‐3 was expressed by 83% of the metastases and in the dermal component of one nodular melanoma. These findings suggest that the loss of AP‐2α protein expression during the progression of melanoma could be related to mutation of the gene in only a small number of tumours, whereas the expression and activation of caspases, most prominently caspase‐6, may be an important factor for the down‐regulation of AP‐2α protein. Furthermore, this study supports recent data that the activation of caspases does not inevitably result in apoptosis, but may also contribute to tumour progression in melanomas. Copyright

A case of a persistent left vena cava superior with atresia of the right atrial ostium of the coronary sinus.

A persistent left vena cava superior with an atretic ostium of the coronary sinus was found during the routine dissecting course in the embalmed cadaver of an 83-year-old woman who had died from cardiac infarction. The left vena cava superior was very narrow in diameter (4 mm), originated at the lateral part of the left vena brachiocephalica and ran down between the venae pulmonales sinistrae and the auricula sinistra. The vena cava opened into the sinus coronarius of the heart, which terminated as a blind sac due to an atretic ostium. The vena coronaria sinistra as well as the vena interventricularis posterior drained into the sinus coronarius. Congenital atresia of the coronary opening is a rare malformation and is usually associated with other anomalies. The congenital ostial atresia could be the cause of a persistent left vena cava superior, which then takes over the drainage of the cardiac veins.

In vivo imaging of the conjunctival epithelium using confocal laser scanning microscopy

ZusammenfassungHintergrundDie zytomorphologische Beurteilung des Oberflächenepithels der Konjunktiva ist bei verschiedenen Augenerkrankungen ein wesentlicher Bestandteil der klinischen Stufendiagnostik. Voraussetzung dafür ist bislang die minimal-invasive Materialentnahme und labortechnische Probenaufbereitung. Für eine zeitnahe direkte In-vivo-Darstellung kornealer Strukturen wird seit geraumer Zeit die konfokale Laser-Scanning-Mikroskopie mit dem Rostocker Laser-Scanning-Mikroskop (RLSM) erfolgreich eingesetzt. Mit einer vergleichenden Untersuchung des normalen Bindehautepithels zwischen der Lichtmikroskopie und der konfokalen RLSM sollen wesentliche Vorraussetzungen für eine In-vivo-Evaluierung des konjunktivalen Epithels geschaffen werden.Material und MethodeFür den intraindividuellen Vergleich wurden 110 impressionszytologische Flächenpräparate von 23 augengesunden Probanden analysiert. Die vergleichende Mustererkennung erfolgte nach Herstellung eines flächenhaften, direkten Kontaktes zwischen der Augenoberfläche und der planen Vorderfläche des RLSM.ErgebnisseAnhand der Verteilung der Reflektoren (helle Bildpunkte) lassen sich bereits in der In-vivo-Diagnostik wichtige morphologische Strukturen des Epithels (Zellkern, Zytoplasma, Kern-Plasma-Relation) differenzieren. Anhand von Form und Größe sind sekretorische Zellen mit dem RLSM zu erfassen. Hochreflektive, helle Bildpunkte stellen kontrastreich die Zellwandung der Becherzellen bzw. breite Intrazellularräume dar.SchlussfolgerungMit dem RLSM gelingt die In-vivo-Untersuchung wesentlicher anatomisch-morphologischer Strukturen des Bindehautepithels. Im Hell-Dunkel-Erkennungsmuster der Becherzellen imponieren Unterschiede, welche auf die Sekretzusammensetzung oder den Funktionszustand (Hypo- oder Hypersekretion) Hinweise geben könnten.AbstractBackgroundIn various ocular diseases, cytomorphological findings of the ocular surface are an essential component of clinical diagnostics. When evaluating the conjunctival epithelium, minimally invasive acquisition of biomaterial is necessary for lab and technical processing and in vitro histological examination. To examine corneal structures in vivo, confocal laser scanning microscopy is a successful standard method. Our aim was to employ in vivo confocal laser scanning microscopy also for examining the conjunctival epithelium.Material and methodResults were analyzed and compared with cytomorphological findings of impression cytology. Accordingly, the basic features of conjunctival in vivo examination using RLSM were described and defined. In vivo images were analyzed and compared with impression cytological slide preparations (n=110) of 23 healthy test persons. Examination was standardized. Finally, the confocal laser scan images were compared to the impression cytological patterns.ResultsDue to the distribution of reflectors (pixel brightness), diagnostic analysis of important morphological structures (cell nucleus, cytoplasm, nucleus/plasma relation) of the conjunctiva is possible. Secretory cells of the epithelium (goblet cells) can be easily recognized by their size. Highly reflective pixels depict cell walls or wide intercellular spaces with high contrast.ConclusionsThe in vivo investigation of important anatomical and morphological structures of the conjunctival epithelium is possible using RLSM. The distribution pattern of goblet cell pixel brightness may correlate with various secretion contents or suggest distinct, recognizable, functional conditions (hypo- or hypersecretion).

In-vivo-Darstellung des Bindehautepithels

ZusammenfassungHintergrundDie zytomorphologische Beurteilung des Oberflächenepithels der Konjunktiva ist bei verschiedenen Augenerkrankungen ein wesentlicher Bestandteil der klinischen Stufendiagnostik. Voraussetzung dafür ist bislang die minimal-invasive Materialentnahme und labortechnische Probenaufbereitung. Für eine zeitnahe direkte In-vivo-Darstellung kornealer Strukturen wird seit geraumer Zeit die konfokale Laser-Scanning-Mikroskopie mit dem Rostocker Laser-Scanning-Mikroskop (RLSM) erfolgreich eingesetzt. Mit einer vergleichenden Untersuchung des normalen Bindehautepithels zwischen der Lichtmikroskopie und der konfokalen RLSM sollen wesentliche Vorraussetzungen für eine In-vivo-Evaluierung des konjunktivalen Epithels geschaffen werden.Material und MethodeFür den intraindividuellen Vergleich wurden 110 impressionszytologische Flächenpräparate von 23 augengesunden Probanden analysiert. Die vergleichende Mustererkennung erfolgte nach Herstellung eines flächenhaften, direkten Kontaktes zwischen der Augenoberfläche und der planen Vorderfläche des RLSM.ErgebnisseAnhand der Verteilung der Reflektoren (helle Bildpunkte) lassen sich bereits in der In-vivo-Diagnostik wichtige morphologische Strukturen des Epithels (Zellkern, Zytoplasma, Kern-Plasma-Relation) differenzieren. Anhand von Form und Größe sind sekretorische Zellen mit dem RLSM zu erfassen. Hochreflektive, helle Bildpunkte stellen kontrastreich die Zellwandung der Becherzellen bzw. breite Intrazellularräume dar.SchlussfolgerungMit dem RLSM gelingt die In-vivo-Untersuchung wesentlicher anatomisch-morphologischer Strukturen des Bindehautepithels. Im Hell-Dunkel-Erkennungsmuster der Becherzellen imponieren Unterschiede, welche auf die Sekretzusammensetzung oder den Funktionszustand (Hypo- oder Hypersekretion) Hinweise geben könnten.AbstractBackgroundIn various ocular diseases, cytomorphological findings of the ocular surface are an essential component of clinical diagnostics. When evaluating the conjunctival epithelium, minimally invasive acquisition of biomaterial is necessary for lab and technical processing and in vitro histological examination. To examine corneal structures in vivo, confocal laser scanning microscopy is a successful standard method. Our aim was to employ in vivo confocal laser scanning microscopy also for examining the conjunctival epithelium.Material and methodResults were analyzed and compared with cytomorphological findings of impression cytology. Accordingly, the basic features of conjunctival in vivo examination using RLSM were described and defined. In vivo images were analyzed and compared with impression cytological slide preparations (n=110) of 23 healthy test persons. Examination was standardized. Finally, the confocal laser scan images were compared to the impression cytological patterns.ResultsDue to the distribution of reflectors (pixel brightness), diagnostic analysis of important morphological structures (cell nucleus, cytoplasm, nucleus/plasma relation) of the conjunctiva is possible. Secretory cells of the epithelium (goblet cells) can be easily recognized by their size. Highly reflective pixels depict cell walls or wide intercellular spaces with high contrast.ConclusionsThe in vivo investigation of important anatomical and morphological structures of the conjunctival epithelium is possible using RLSM. The distribution pattern of goblet cell pixel brightness may correlate with various secretion contents or suggest distinct, recognizable, functional conditions (hypo- or hypersecretion).

Loss of heterozygosity at 12p13 and loss of p27KIP1 protein expression contribute to melanoma progression.

Little is known about the mechanisms causing p27KIP1 decrease in melanomas. Therefore, we performed loss of heterozygosity (LOH) analysis with polymerase chain reaction at seven different loci surrounding the p27KIP1/CDKN1B gene at 12p13 and direct DNA sequencing analysis of all exons. Furthermore, the immunohistochemical expression of p27KIP1 and Ki-67 was investigated. Only two mutations in the sequence of p27KIP1/CDKN1B were detected, but the number of tumours showing LOH at 12p13 increased significantly with the parameters of tumour progression (pT level, P=0.018; Breslow index, P=0.01; Clark level, P<0.001), with a more aggressive tumour growth (radial versus vertical growth, P=0.018) and tumour subtype (superficial spreading melanomas versus nodular melanomas versus metastases, P<0.001). p27KIP1 protein expression decreased with the Clark level (P=0.026) and the pT level (P=0.045). No correlation between LOH affecting 12p13 and p27KIP1 protein decrease in melanomas was stated. This does not exclude the participation of p27KIP1/CDKN1B in p27KIP1 protein decrease, since protein expression is regulated at various cellular levels; but it could also suggest that other tumour suppressors are situated in the same region as p27KIP1/CDKN1B. Taken together, our data shows that loss of p27KIP1 protein expression and LOH at 12p13 contribute to tumour progression in melanoma.

Dominance of cytokine- over FasL-induced impairment of the mitochondrial transmembrane potential (ΔΨm) in the pancreatic β-cell line NIT-1

Mitochondria of pancreatic β-cells are potential targets of intrinsic and extrinsic apoptotic pathways in the autoimmune pathogenesis of type 1 diabetes. We aimed to investigate whether cytokine- and FasLigand (FasL)-induced apoptosis is associated with impaired mitochondrial transmembrane potential (ΔΨm) in the pancreatic β-cell line NIT-1. NIT-1 cells were exposed to the interleukin-1 β/interferon-γ (IL-1 β/IFN-γ) cytokine combination to induce apoptosis in vitro. Low concentrations of cytokines resulted in ΔΨm impairment, and increasing concentrations had only a minor additional effect. Treatment with the inducible nitric oxide synthase (iNOS) inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) prevented cytokine-mediated ΔΨm impairment, implying that cytokines affect ΔΨm via nitric oxide. The broad-spectrum caspase inhibitor Z-VAD(Ome)-FMK (ZVAD) revealed dichotomic actions. In the presence of ZVAD, cytokine-induced nitrite generation was increased but cell death and ΔΨm impairment were reduced. ΔΨm impairment was also reduced by inhibitors of caspases 1, 6 and 8. Induction of Fas by IL-1 β/IFN-γ coupled with activation by Super-FasL augmented cytokine-induced cell death. We observed a clear dominance of cytokine- over FasL-induced effects on ΔΨm. Our findings show that IL-1 β/IFN-γ cytokines have a strong effect to impair ΔΨm and prime β-cells for apoptosis via the intrinsic pathway mediated by iNOS and caspases. Furthermore, at least in NIT-1 cells, the extrinsic FasL/Fas pathway has only a minor additive effect on cytokine-induced ΔΨm impairment.