Csaba Mahotka

Survivin splice variants regulate the balance between proliferation and cell death.

Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-ΔEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.

Differential subcellular localization of functionally divergent survivin splice variants.

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-ΔEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-ΔEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-ΔEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-ΔEx3 may support cytoplasmic clearance of survivin-ΔEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-ΔEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.

Expression of different survivin variants in gastric carcinomas: first clues to a role of survivin-2B in tumour progression

Survivin is a novel member of the inhibitor of apoptosis family and determines the susceptibility of tumour cells to pro-apoptotic stimuli. Recently, we identified two novel alternative splice variants of survivin, differing in their anti-apoptotic properties: whereas the anti-apoptotic potential of survivin-ΔEx3 is preserved, survivin-2B has lost its anti-apoptotic potential and may act as a naturally occurring antagonist of survivin. Because the in vivo expression of these alternative splice variants has not been explored so far, we analysed gastric carcinomas of different histological subtypes, grades and stages. Since no antibodies are currently available to determine the novel splice variants, quantitative reverse transcriptase polymerase chain reaction was performed, using RNA samples obtained from 30 different gastric carcinomas. Polymerase chain reactions products were quantified by densitometric evaluation. We found that all gastric carcinomas, irrespective of their histological types, grades or stages, express survivin-ΔEx3, survivin-2B and survivin, the latter being the dominant transcript. Comparing the disease stages I+II with III+IV, expression of survivin and survivin-ΔEx3 remained unchanged. In contrast, a significant (P=0.033) stage-dependent decrease in the expression of survivin-2B became evident. Our study demonstrates for the first time the expression of alternative splice variants in gastric carcinomas and provides a first clue to a role of survivin-2B in tumour progression.

Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas

Survivin, a novel member of the inhibitor of apoptosis protein (IAP) family, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor progression and treatment with anticancer drugs. Recently, we identified 2 novel alternative splice variants of survivin, survivin‐2B and survivin‐ΔEx3, which differ in their antiapoptotic properties. Survivin‐2B has lost its antiapoptotic potential and may act as a naturally occurring antagonist of antiapoptotic survivin and survivin‐ΔEx3. Because the in vivo expression of these splice variants in human cancer has not been analyzed so far, 57 renal cell carcinomas (RCCs) were explored using quantitative reverse transcriptase polymerase chain reaction. We found that all RCCs express survivin‐ΔEx3, survivin‐2B and survivin, the latter being the dominant transcript. When we compared early and intermediate stages with late stages of clear cell RCCs, no significant changes in the expression levels of survivin and survivin‐ΔEx3 became evident. However, a significant decrease was observed for the mRNA ratio between survivin‐2B and survivin in late tumor stages (p = 0.036). Chromophilic/papillary RCCs, which are known to be less aggressive than clear cell RCCs, did not show significantly lower expression levels of antiapoptotic survivin and survivin‐ΔEx3, compared with stage‐adjusted clear cell RCCs. Our study demonstrates for the first time in vivo expression of functionally different survivin variants and suggests a role of these survivin splice variants in the progression and clinical behavior of human RCCs.

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.

Sensitivity to TRAIL/APO-2L-mediated apoptosis in human renal cell carcinomas and its enhancement by topotecan

TRAIL (APO-2L) is a newly identified member of the TNF family and induces apoptosis in cancer cells without affecting most non-neoplastic cells, both in vitro and in vivo. Our study focused on the expression and function of TRAIL and its receptors in renal cell carcinoma (RCC) cell lines of all major histological types. Here, we demonstrate that all RCC cell lines express TRAIL as well as the death-inducing receptors TRAIL-R1 (DR4) and TRAIL-R2 (Killer/DR5). Exposure to TRAIL induced apoptosis in 10 of 16 RCC cell lines. Remarkably, five of six TRAIL-resistant RCC cell lines exhibited high levels of TRAIL expression. Topotecan, a novel topoisomerase I inhibitor, induced upregulation of TRAIL-R2 as well as downregulation of TRAIL. Neutralization of TRAIL with recombinant soluble TRAIL-R1-Fc and TRAIL-R2-Fc failed to inhibit topotecan-induced apoptosis indicating that topotecan-induced cell death can occur in a TRAIL-independent fashion. However, exposure to topotecan resulted in an enhancement of TRAIL-induced apoptosis in all primarily TRAIL-resistant RCC cell lines. This synergistic effect of cotreatment with Topotecan and TRAIL may provide the basis for a new therapeutic approach to induce apoptosis in otherwise unresponsive RCC. Cell Death and Differentiation (2000) 7, 1127–1136.

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.

DECREASE OF DNA METHYLTRANSFERASE 1 EXPRESSION RELATIVE TO CELL PROLIFERATION IN TRANSITIONAL CELL CARCINOMA

In many common cancers such as transitional cell carcinoma (TCC), specific genes are hypermethylated, whereas overall DNA methylation is diminished. Genome‐wide DNA hypomethylation mostly affects repetitive sequences such as LINE‐1 retrotransposons. Methylation of these sequences depends on adequate expression of DNA methyltransferase I (DNMT1) during DNA replication. Therefore, DNMT1 expression relative to proliferation was investigated in TCC cell lines and tissue as well as in renal carcinoma (RCC) cell lines, which also display hypomethylation, as indicated by decreased LINE‐1 methylation. Cultured normal uroepithelial cells or normal bladder tissue served as controls. In all tumor cell lines, DNMT1 mRNA as well as protein was decreased relative to the DNA replication factor PCNA, and DNA hypomethylation was present. However, the extents of hypomethylation and DNMT1 downregulation did not correlate. Reporter gene assays showed that the differences in DNMT1 expression between normal and tumor cells were not established at the level of DNMT1 promoter regulation. Diminished DNMT1:PCNA mRNA ratios were also found in 28/45 TCC tissues but did not correlate with the extent of DNA hypomethylation. In addition, expression of the presumed de novo methyltransferases DNMT3A and DNMT3B mRNAs was investigated. DNMT3B overexpression was observed in about half of all high‐stage TCC (DNMT3B vs. tumor stage, χ2: p = 0.03), whereas overexpression of DNMT3A was rarer and less pronounced. Expression of DNMT3A and DNMT3B in most RCC lines was higher than in TCC lines. Our data indicate that DNMT1 expression does not increase adequately with cell proliferation in bladder cancer. This relative downregulation probably contributes to hypomethylation of repetitive DNA but does not determine its extent alone.

The presence of nitrite during UVA irradiation protects from apoptosis

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration‐dependent protection against UVA‐induced apoptotic cell death. Protection is half‐maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite‐ mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite‐ mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA‐induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA‐induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

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.