Uwe Ramp

Diagnostic accuracy of effusion cytology

The aim of this investigation was to report on the diagnostic accuracy of conventional effusion cytology. Cytological diagnoses of 300 pleural effusions and 300 ascites were compared with clinical and/or histological follow‐ups of the respective patients. Sensitivity of our cytological diagnoses on pleural effusions was 50.0%, specificity 97.0%, positive predictive value 95.7%, and negative predictive value 86.4%. Sensitivity in ascitic effusions was 62.4%, specificity 98.0%, positive predictive value 100.0%, and negative predictive value 88.3%; 5.8% of diagnoses for pleural and 4.4% for peritoneal effusions were suspicious or doubtful. The overall false‐positive rate was 0.5%, while the false‐negative rate was 31.5%. False‐negative results were due to sampling errors in 71% of pleural and 73% of peritoneal effusions and to screening errors in 29% and 27%, respectively. Our data and those from the literature show that diagnostic accuracy of effusion cytology is still unsatisfactory and should be improved. Therefore, the use of different adjuvant methods is recommended. Diagn. Cytopathol. 1999;20:350–357.

Disturbed balance of expression between XIAP and Smac/DIABLO during tumour progression in renal cell carcinomas

Dysregulation of apoptosis plays an important role in tumour progression and resistance to chemotherapy. The X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent caspase inhibitor of all known inhibitor of apoptosis-family members. Only recently, an antagonist of XIAP has been identified, termed Smac/DIABLO. To explore the relevance of antiapoptotic XIAP and proapoptotic Smac/DIABLO for tumour progression in renal cell carcinomas (RCCs), we analysed XIAP and Smac/DIABLO mRNA and protein expression in the primary tumour tissue from 66 RCCs of all major histological types by quantitative real-time PCR, Western blot and ELISA. X-linked inhibitor of apoptosis and Smac/DIABLO mRNA expression was found in all RCCs. Importantly, the relative XIAP mRNA expression levels significantly increased from early (pT1) to advanced (pT3) tumour stages (P=0.0002) and also with tumour dedifferentiation (P=0.04). Western blot analysis confirmed the tumour stage-dependent increase of XIAP expression on the protein level. In contrast, mRNA and protein expression levels of Smac/DIABLO did not significantly change between early and advanced tumour stages or between low and high tumour grades. Consequently, the mRNA expression ratio between antiapoptotic XIAP and proapoptotic Smac/DIABLO markedly increased during progression from early (pT1) to advanced (pT3) tumour stages. Moreover, RCCs confined within the organ capsule (pT1 and pT2) exhibited a significantly lower XIAP to Smac/DIABLO expression ratio when compared with RCCs infiltrating beyond the kidney (pT3; P=0.01). Thus, our investigation demonstrates that the delicate balance between XIAP and Smac/DIABLO expression is gradually disturbed during progression of RCCs, resulting in a relative increase of antiapoptotic XIAP over proapoptotic Smac/DIABLO, thereby probably contributing to the marked apoptosis resistance of RCC.

Apoptosis induction in renal cell carcinoma by TRAIL and γ -radiation is impaired by deficient caspase-9 cleavage

TNF-related apoptosis-inducing ligand (TRAIL APO-2L) is a member of the TNF family and induces apoptosis in cancer cells without affecting most non-neoplastic cells. The present investigation is focused on apoptosis induction by combined exposure to TRAIL and ionising radiation (IR) in human renal cell carcinoma (RCC) cell lines. Here, we demonstrate that all RCC cell lines coexpress TRAIL and the death-inducing receptors, TRAIL-R1 and TRAIL-R2. Exposure to TRAIL alone induced marked apoptosis in three out of eight RCC cell lines. Combined exposure to TRAIL and IR resulted in a sensitisation to TRAIL-induced apoptosis in one RCC cell line only. Enhanced apoptosis induction by TRAIL in combination with IR was paralleled by an increase in PARP cleavage and activation of executioner caspase-3, whereas caspases-6 and -7 were not involved. Moreover, exposure to TRAIL and/or IR resulted in a marked activation of initiator caspase-8, possibly augmented by the observed reduction of inhibitory c-FLIP expression. In contrast to other tumour types, activation of initiator caspase-9 was not detectable in our RCC model system after exposure to TRAIL and/or IR. This lack of caspase-9 activation might be related to an impaired ‘crosstalk’ with the caspase-8 pathway as suggested by the missing Bid cleavage and to the appearance of an XIAP cleavage product known to inhibit caspase-9 activation. Deficient activation of caspase-9, therefore, might contribute to the clinically known resistance of human RCC against IR and also argues against an effective combination therapy with TRAIL and IR in this tumour type.

TRAIL-β and TRAIL-γ: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential

Tumour necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL/APO2L) is a recently identified member of the TNF family, which induces programmed cell death in a variety of neoplastic cell types, but not in most nonneoplastic cells. In this study, we report on the identification of two novel alternative splice variants of TRAIL in neoplastic and non-neoplastic human cells lacking either exon 3 (TRAIL-β) or exons 2 and 3 (TRAIL-γ). In both splice variants, loss of exon 3 resulted in a frame shift generating a stop codon with consecutive extensive truncation in the extracellular domain. Ectopic expression revealed a loss of proapoptotic potential for both alternative splice variants. In contrast to the predominantly cytoplasmatic localisation of GFP-tagged TRAIL-α and TRAIL-β, TRAIL-γ showed an additional association with the cell surface and nuclear membrane. In conclusion, alternative splicing might be involved in fine tuning of TRAIL-induced apoptosis and underlines the complexity of the TRAIL system.

Deficient activation of CD95 (APO-1/ Fas)-mediated apoptosis: a potential factor of multidrug resistance in human renal cell carcinoma

The pronounced resistance of human renal cell carcinoma (RCC) to anticancer-induced apoptosis has primarily been related to the expression of P-glycoprotein and effective drug detoxification mechanisms. Because the CD95 system has recently been identified as a key mediator of anticancer drug-induced apoptosis, we analysed the contribution of the CD95 system to chemotherapy-induced apoptosis in four newly established RCC cell lines. Here, we demonstrate that all RCC cell lines expressed CD95-receptor and -ligand. Exposure to agonistic anti-CD95 antibodies resulted in induction of apoptosis and significant (P< 0.05) reduction of cell number in three out of four cell lines, indicating that the essential components for CD95-mediated apoptosis were present and functionally intact in the majority of these RCC cell lines. Moreover, treatment of cultures with bleomycin or topotecan, a novel topoisomerase I inhibitor with little substrate affinity for P-glycoprotein, led to induction of apoptosis and significant (P< 0.05) dose-dependent reduction of cell number in all RCC cell lines. Both anticancer drugs also induced upregulation of CD95 ligand expression in all cell lines. Additionally, augmentation of CD95 receptor expression was found in three RCC cell lines, including one p53-mutated cell line, whereas another p53-mutated cell line showed no or only a weak CD95 receptor upregulation after exposure to topotecan or bleomycin, respectively. Despite this upregulation of CD95 receptor and ligand, antagonistic antibodies directed against CD95 receptors or ligands could not inhibit induction of apoptosis by topotecan and bleomycin in any cell line. Thus, although a functionally intact CD95 signalling cascade is present in most RCC cell lines, the anticancer drugs topotecan and bleomycin that induce upregulation of CD95 receptor and ligand fail to effectively activate CD95-mediated apoptosis. This deficient activation of CD95-mediated apoptosis might be an important additional factor for the multidrug resistance phenotype of human RCCs.

Functional Intactness of Stimulatory and Inhibitory Autocrine Loops in Human Renal Carcinoma Cell Lines of the Clear Cell Type

PURPOSEnThe aim of the present study was to analyze the contribution of different stimulatory and inhibitory growth factors to the deregulated proliferation of human RCCs.nnnMATERIALS AND METHODSnThe expression of different growth factors and their corresponding receptors were analyzed by Northern blot, FACS, ELISA and immunocytochemistry in 13 permanent human RCC cell lines of the clear cell type. Moreover, the functional intactness of growth factor-related signal transduction pathways was investigated.nnnRESULTSnAll RCC cell lines expressed EGF-receptor mRNA and protein and 10 cell lines secreted TGF-alpha. Exogeneously added TGF-alpha resulted in a significant (p < 0.05) stimulation of growth in 6 RCC cell lines and a significant (p < 0.05) inhibition of proliferation in 3 cell lines. PDGF B and the corresponding type beta receptor were expressed in a single cell line. mRNA expression of PDGF A and PDGF-alpha-receptor as well as IGF-1 and its receptor could not be detected in any cell line. Eleven RCC cell lines expressed TGF-beta 1 mRNA and in all cell lines TGF-beta 1 secretion into the supernatant could be demonstrated. Whereas all cell lines exhibited TGF-beta type II-receptor mRNA, type I-receptor mRNA could be detected only in 3 cell lines. TGF-beta type III-receptor was observed in 1 cell line. Exogeneously added TGF-beta1 resulted in a significant (p < 0.05) inhibition of proliferation in 7 RCC cell lines.nnnCONCLUSIONnClear cell RCCs exhibit a complex and heterogeneous expression pattern for various growth factors and their receptors. Growth factor secretion and intact signal transduction pathways in most clear cell RCCs facilitate an intricate modulation of RCC growth by autocrine and paracrine interactions between tumor cells and host tissue.

Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC

Activation of the initiator-caspase, caspase-8 is under tight control of multiple antiapoptotic regulators including ARC, cFlipS, cFlipL and PED/PEA-15. Since there is little data regarding the expression of caspase-8 and its antiapoptotic regulators in human tumours inxa0vivo, we analysed their expression in renal cell carcinomas (RCCs) to identify which of these genes might be crucial for the well known impaired apoptosis and—as a result—resistance towards chemotherapy and ionizing radiation of RCCs. Caspase-8, cFlipS, cFlipL and PED/PEA-15 mRNA expression was significantly increased only in early stages of RCCs compared to non-neoplastic renal tissue. In contrast, ARC mRNA expression was significantly increased in RCCs of all stages without differences between the tumour stages and grades. Importantly, the relative mRNA expression ratio between ARC and caspase-8 was significantly increased during carcinogenesis and tumour progression. In contrast, the relative mRNA expression ratio between cFlipS, cFlipL or PED/PEA-15 and caspase-8 remained constant during all tumour stages. In conclusion, our analysis revealed that ARC is the only caspase-8 inhibiting regulator being constantly overexpressed in RCCs. Furthermore, the balance between antiapoptotic ARC and proapoptotic caspase-8 is the only one to be disturbed during carcinogenesis and tumour progression of RCCs. This inhibition of Caspase-8 might therefore be one example for the multiple antiapoptotic functions of ARC in RCCs possibly contributing to the marked resistance of RCCs towards radio- and chemotherapy and reflects a shift of gene expression towards a more antiapoptotic context in RCCs.

Disturbed XIAP and XAF1 Expression Balance Is an Independent Prognostic Factor in Gastric Adenocarcinomas

BackgroundDysregulation of apoptosis is a key factor in carcinogenesis and tumor progression. X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) family, which directly inhibits apoptosis by binding to caspases. Antagonists of XIAP have recently been identified: second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI (Smac/DIABLO) and XIAP-associated factor 1 (XAF1). However, little research has been conducted on the association between gastric cancer survival and the mechanism of apoptosis involving XIAP and its antagonists, Smac/DIABLO and XAF1.MethodsXIAP, Smac/DIABLO, and XAF1 expression was analyzed by immunohistochemistry (IHC) in 187 gastric adenocarcinomas. Correlations between XIAP, Smac/DIABLO or XAF1 expression and clinicopathological factors were analyzed. Disease-specific survival after surgery was examined.ResultsOf 187 samples, XIAP was overexpressed in 140, Smac was overexpressed in 117, and XAF1 was overexpressed in 106. Individually, XIAP, Smac, and XAF1 were not significantly associated with disease-specific survival. However, patients showing high expression of XIAP and low expression of XAF1 had significantly poorer survival when compared with other groups (Pxa0=xa00.024).ConclusionThe expression balance of XIAP and XAF1 is an independent prognostic factor in gastric adenocarcinoma.

Disturbed expression of the apoptosis regulators XIAP, XAF1, and Smac/DIABLO in gastric adenocarcinomas.

Dysregulation of apoptosis plays an important role in carcinogenesis and tumor progression. Whereas x-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of apoptosis, its antagonists second mitochondria-derived activator of caspases/direct IAP binding protein with low PI (Smac/DIABLO), and XIAP-associated factor 1 (XAF1) promote apoptosis. To explore the relevance of XIAP, Smac/DIABLO, and XAF1 for carcinogenesis and tumor progression, we analyzed 46 primary gastric adenocarcinomas and non-neoplastic gastric mucosa samples by quantitative real-time polymerase chain reaction. XIAP, Smac/DIABLO, and XAF1 expression was found in all non-neoplastic gastric mucosa samples and all adenocarcinomas. XIAP expression levels did not change between non-neoplastic gastric mucosa and adenocarcinomas or between carcinomas of early and advanced stages. Although Smac/DIABLO expression was significantly (P=0.01) higher in carcinomas, the ratio of XIAP to Smac/DIABLO expression remained stable between non-neoplastic mucosa and carcinomas. XAF1 expression had the tendency to decrease from non-neoplastic mucosa to advanced adenocarcinomas. Importantly, the ratio of XIAP to XAF1 expression significantly (P=0.03) increased from non-neoplastic mucosa to adenocarcinomas and the increase was even higher in carcinomas of advanced stage (P=0.01). Moreover, expression of the XAF1 splice variants differing in the zinc-finger domain essential for XIAP-binding was analyzed and revealed a significant higher (P=0.03) variant-2/variant-1 ratio in advanced carcinomas. In conclusion, an increased expression ratio of XIAP to XAF1 in combination with a disturbed expression of the XAF1 splice variants could be shown in gastric adenocarcinomas. These marked imbalances probably result in an impaired ability for XAF1 to antagonize the effects of XIAP thereby contributing to apoptosis-resistance and generating an important growth advantage.

Differential response to transforming growth factor (TGF)-α and fibroblast growth factor (FGF) in human renal cell carcinomas of the clear cell and papillary types

The clear cell and the papillary types of human renal cell carcinoma (RCC) are distinct tumour entities with marked differences in their biological properties. Because growth factors are considered to affect profoundly the biological behaviour of malignant tumours, we compared the expression and function of transforming growth factor (TGF)-alpha and fibroblast growth factor (FGF) in both types of RCCs. Both in vivo and in vitro expression of TGF-alpha, epidermal growth factor-receptor (EGF-R), FGF-2 and FGF type 3- and 4-receptors was found in RCCs of both types. However, marked differences between clear cell and papillary RCCs became evident for TGF-alpha secretion, which could be demonstrated in 20 out of 24 (83%) clear cell RCCs but in only two out of four (50%) papillary tumours. Moreover, the mean TGF-alpha secretion rate in clear cell RCCs significantly (P<0. 05) exceeded that of papillary RCCs. Because the expression of growth factor receptors could not prove the corresponding signalling cascades were functional, tumour cell proliferation was tested after exposure to exogenous TGF-alpha or FGF-1. These experiments demonstrated that papillary RCCs did not respond significantly to exogenous TGF-alpha or FGF-1, whereas eight (33%) (TGF-alpha) and 11 (46%) (FGF-1) out of 24 clear cell RCCs responded with significant (P<0.05) growth stimulation. In conclusion, our investigation presents data indicating that TGF-alpha and FGF are functionally involved in the progression of clear cell RCCs, directly stimulating proliferation by autocrine and/or paracrine actions. In contrast, TGF-alpha and FGF did not directly stimulate the proliferation of our papillary RCCs, thereby suggesting functional defects or a blockade in the corresponding signalling cascades. This differential functionality might contribute to the more aggressive behaviour of clear cell RCCs.