Jens Mollerup

Up-regulation of ALG-2 in hepatomas and lung cancer tissue.

ALG-2 was isolated in a screen for proteins involved in programmed cell death and is the first Ca(2+)-binding protein found to be directly involved in apoptosis. We have generated polyclonal antibodies that are suitable for detecting ALG-2 using different immunological methods. Three commercial antibodies against ALG-2 did neither detect mouse recombinant ALG-2 nor endogenous ALG-2 in Jurkat cell lysates, whereas our own affinity-purified antibody recognized recombinant as well as endogenous ALG-2. The specificity of the antibody was shown by preabsorbtion experiments and on ALG-2-deficient cells using Western blot analysis and immunohistochemistry. Western blot analysis of 15 different adult mouse tissues demonstrated that ALG-2 is ubiquitously expressed. We found that ALG-2 was more than threefold overexpressed in rat liver hepatoma compared to normal rat liver using Western blot analysis, a result confirmed by immunohistochemical analysis. Staining of four different lung cancer tissue microarrays including specimens of 263 patients showed that ALG-2 is mainly localized to epithelial cells and significantly up-regulated in small-cell lung cancers and in non-small-cell lung cancers. Our results lead to the conclusion that ALG-2 beside its known proapoptotic functions may be a player in survival pathways.

The apoptosis linked gene ALG-2 is dysregulated in tumors of various origin and contributes to cancer cell viability

The apoptosis linked gene‐2 (ALG‐2), discovered as a proapoptotic calcium binding protein, has recently been found upregulated in lung cancer tissue indicating that this protein may play a role in the pathology of cancer cells and/or may be a tumor marker. Using immunohistochemistry on tissue microarrays we analysed the expression of ALG‐2 in 7371 tumor tissue samples of various origin as well as in 749 normal tissue samples. Most notably, ALG‐2 was upregulated in mesenchymal tumors. No correlation was found between ALG‐2 staining intensity and survival of patients with lung, breast or colon cancer. siRNA mediated ALG‐2 downregulation led to a significant reduction in viability of HeLa cells indicating that ALG‐2 may contribute to tumor development and expansion.

ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death.

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.

Properties of the co-chaperone protein p23 erroneously attributed to ALG-2 (apoptosis-linked gene 2).

A commercial antibody (clone 22) directed against the apoptosis‐linked gene 2 (alg2, pdcd6) encoded protein has been used by several groups. Up‐regulated expression of the antigen was observed in primary tumours and in metastatic tissue and also during rat brain ischemia. Furthermore, antigen down‐regulation was found in human atherosclerotic plaques. Recently, we found that the clone 22 antibody does not recognise ALG‐2. In the present study the antigen of the clone 22 antibody was identified as the heat shock protein 90 (HSP90) co‐chaperone protein p23, identical to the cytosolic prostaglandin E2 synthase, by immunoprecipitation followed by tryptic in‐gel digests and mass spectrometry of the purified peptides. Moreover, the heterogeneous ribonuclear protein A2/B1 was found to be a part of the p23 co‐immunoprecipitated protein complex.

The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis.

The heat shock protein 90 co‐chaperone p23 has recently been shown to be up‐regulated in cancer cells and down‐regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFα‐receptor activation as well as staurosporine treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase‐3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18 kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis. Furthermore, we found that the anti‐aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis.

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.

Calyculin A modulates the kinetic constants for the Na+-coupled taurine transport in Ehrlich ascites tumour cells

The effect of the phosphatase inhibitor calyculin A (cal A) on the kinetic parameters of the Na+-coupled taurine uptake via the taurine transporter in the Ehrlich ascites tumour cells has been investigated. Preincubation with cal A (100 nM) reduces the initial taurine influx by about 20%, but has no effect on the diffusional component of the taurine influx or on the taurine release from cells suspended in isotonic or in hypotonic medium. Thus, cal A-sensitive phosphatases only affect taurine transport mediated by the Na+-dependent taurine transporter. Cal A increases the Michaelis-Menten constant for binding of taurine to the transporter from 31+/-6 to 45+/-4 microM and reduces the taurine transport capacity from 210+/-20 to 170+/-10 nmol x g dry wt(-1) x min(-1) [corrected]. The Michaelis-Menten constant for binding of Na+ to the taurine transporter is concomitantly increased from 96+/-11 to 129+/-8 mM and the Na+:taurine coupling ratio for activation of the transport cycle is reduced from 3.3+/-0.6 to 2.4+/-0.2. This suggests that cal A-sensitive phosphatases maintain a high affinity of the taurine transporter towards Na+ and taurine as well as a high taurine transport capacity in unpertubated Ehrlich cells.

Homologous desensitisation of the mouse leukotriene B4 receptor involves protein kinase C-mediated phosphorylation of serine 127.

Murine leukotriene B₄ (LTB₄) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB₄ receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB₄-evoked, Ca²⁺-activated Cl^- currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca²⁺-activated Cl^- current and LTB₄ concentration was demonstrated with an apparent EC₅₀ of 6.7 nM. Following LTB₄ stimulation of mBLT1, we observed two transient, spatially distinct Ca²⁺-activated, inwardly directed Cl^- currents in the oocytes: a fast peak current requiring relatively high LTB₄ concentrations, and a slowly progressing Cl^- current. Nucleotides, PGE₂, 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD₄ did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB₄-evoked Cl^- currents. Repetitive LTB₄ administration desensitized the LTB₄-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB₄-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.