Radiation therapy for the elderly-change of concepts in breast cancer?

The incidence of breast cancer in elderly patients has increased in the last years, perhaps due to better and earlier diagnostics and a longer life expectancy. Radiotherapy as a local adjuvant treatment after breast surgery, improves local control and overall survival. Due to frailty or comorbidity current guidelines cannot always be applied and the treatment decision-making process involving these older patients often remains the task of the treating physician rather than a fully established tumor board. The goal of this review is to summarize in short, the radiation therapy techniques available, and to outline the most important factors that could be relevant in the decision making for radiotherapy for elderly woman affected by invasive breast cancer.

Possible predictors of histopathological response to neoadjuvant chemoradiotherapy for rectal cancer.

PURPOSE: The response to neoadjuvant chemoradiotherapy (CRT) varies greatly in patients suffering from locally advanced rectal cancer. Our aim was to correlate the response to CRT with the pre-treatment expression of heat shock protein 90 (Hsp90), small heat shock protein 16.2 (sHsp 16.2), phospho-Akt (p-Akt), growth hormone-releasing hormone receptor (GHRH-R) and heme-binding protein 2 (SOUL) in order to try to identify one or more as a predictive marker. MATERIALS AND METHODS: Sixty-nine patients receiving combined CRT for locally advanced rectal cancer were examined retrospectively. Surgical resection was carried out 6-9 weeks following CRT. The histopathological response to neoadjuvant treatment was determined according to the modified Mandard score. Using immunohistochemistry, we investigated the relationship between the expression of the five cited proteins in the pre-operative samples as well as various clinical parameters and the histopathological regression of the tumors. RESULTS: Thirty-one patients (48%) were good responders, and 33 patients (52%) were found to respond poorly to neoadjuvant therapy. Among patients undergoing surgery 7 weeks following CRT, there were significantly more good responders than among patients who underwent surgery sooner (63% vs. 37%). High levels of expression of GHRH-R and Hsp90 were shown to be significantly correlated with minor or absent histological regression. CONCLUSIONS: GHRH-R and Hsp90 were found to be independent predictive factors of histopathological response to neoadjuvant RCT. Since GHRH-R antagonists and Hsp90 inhibitors are currently being tested as potential anticancer agents, our study implies the possible elaboration of an effective and individualized treatment of poor responders.

[Modern three-dimensional conformal craniospinal radiotherapy]. / Modern három dimenziós konformális craniospinalis besugárzási technika.

The main problem of craniospinal irradiation (CSI) is the matching of the fields. The use of a suitable technique is very important because matching of the fields is necessary to use for the optimal cancer irradiation of the long planning target volume (PTV). Since 2007, 8 patients have received CT-based, 3D-planned conformal CSI in our Institute. Patient immobilization was made in prone position in a vacuum bed, using skull and pelvis masks. Organ-at-risk (OAR) contours were made by radiographers. PTV was contoured by radiation oncologists. The prescribed dose to the PTV was 36 Gy with 1.8 Gy dose per fraction. In the planning process the following aspects were taken under consideration: all points of the PTV had to receive at least 95% of the prescribed dose (according to ICRU 50, 62); at junction field edges the overlapping parts were eliminated using a multisegmental technique, where the adjacent segment ends of the neighbouring fields were shifted two times 2 cm, so that the three equally weighted segments used in one field had 2-2 cm distance from each other. In the CSI planning the shape of the patient and so the length of the PTV has made a big emphasis on determining the number of field matching. Thus in some cases instead of two, only one field matching was sufficient - this could be achieved by increasing the source-to-skin distance (SSD) of the fields. The verification made with a solid-water phantom justified the precision of the field matching. The offset used at junction field edges in between one treatment facilitates the verification of field matching - and so the patient positioning. Thus the possibility of having overdosed regions could be reduced, which was very important from a radiation biological point of view.

[Conformal stereotactic radiosurgery therapy: plan evaluation methods and results]. / Konformális sztereotaxiás együléses sugársebészeti kezelés: tervértékelési módszerek és eredmények.

The purpose of our study was the objective evaluation of micro-multileaf collimator (mMLC)-based stereotactic radiosurgery treatment plans. Forty-seven patients, 71 lesions received static beam conformal stereotactic radiosurgery treatment in our institute between November 2005 and June 2008. Target volume and organs at risk were outlined on a MRI-CT image fusion basis. BrainSCAN 5.31 system (BrainLAB AG, Heimstetten, Germany) was used for treatment planning, Elekta Presice TS linear accelerator (Elekta Oncology Systems Ltd, Crawley, UK) and BrainLAB m3 mMLC were used for treatment delivery. An invasive head frame, mounted to the treatment table, was used with four screws for patient head fixation. Treatment plans were analysed with objective parameters, such as conformal index (COIN), homogeneity index (HI), coverage index (CI) and healthy tissue relative overdose factor (HTOF) tools. x2 tests were performed between COIN, HI and the geometrical parameters of the target volume (lesion volume - LV, lesion-organ distance - LOD, lesion deformity index - LDI). Mean value of COIN, HI, HTOF and CI was 0.52 (SD 0.13), 1.16 (SD 0.1), 0.88 (SD 0.53), and 0.94 (SD 0.11), respectively. COIN significantly correlated with (p<0.001 in all three cases), while HI was independent of LV, LOD, LDI (p=0.94; 0.14 and 0.72). COIN is similar, HTOF is less than data from the literature. According to our results geometrical parameters of the target volume (size, location, deformation) significantly influence the COIN, but they have no effect on HI.

Conkiss: conformal kidneys sparing 3D noncoplanar radiotherapy treatment for pancreatic cancer as an alternative to IMRT.

When treating pancreatic cancer using standard (ST) 3D conformal radiotherapy (3D-CRT) beam arrangements, the kidneys often receive a higher dose than their probable tolerance limit. Our aim was to elaborate a new planning method that--similarly to IMRT--effectively spares the kidneys without compromising the target coverage. Conformal kidneys sparing (CONKISS) 5-field, noncoplanar plans were compared with ST plans for 23 consecutive patients retrospectively. Optimal beam arrangements were used consisting of a left- and right-wedged beam-pair and an anteroposterior beam inclined in the caudal direction. The wedge direction determination (WEDDE) algorithm was developed to adjust the adequate direction of wedges. The aimed organs at risk (OARs) mean dose limits were: kidney <12 Gy, liver <25 Gy, small bowels <30 Gy, and spinal cord maximum <45 Gy. Conformity and homogeneity indexes with z-test were used to evaluate and compare the different planning approaches. The mean dose to the kidneys decreased significantly (p < 0.05): left kidney 7.7 vs. 10.7 Gy, right kidney 9.1 vs. 11.7 Gy. Meanwhile the mean dose to the liver increased significantly (18.1 vs. 15.0 Gy). The changes in the conformity, homogeneity, and in the doses to other OARs were not significant. The CONKISS method balances the load among the OARs and significantly reduces the dose to the kidneys, without any significant change in the conformity and homogeneity. Using 3D-CRT the CONKISS method can be a smart alternative to IMRT to enhance the possibility of dose escalation.

A pelvic phantom for modeling internal organ motions.

A pelvic phantom was developed for use in testing image-guided radiation therapy (IGRT) and adaptive applications in radiation therapy (ART) with simulating the anterior-posterior internal organ motions during prostate radiotherapy. Measurements could be done with an ionization chamber (IC) in the simulated prostate. The rectum was simulated by air-equivalent material (AEM). The volume superior to the IC placement was considered as the bladder. The extension of AEM volume could be varied. The vertical position of the IC placement could be shifted by ± 1 cm to simulate the prostate motion parallel to the changes in bladder volume. The reality of the simulation was inspected. Three-millimeter-slice-increment computed tomography (CT) scans were taken for irradiation planning. The structure set was adapted to the phantom from a treated patient. Planning target volume was delineated according to the RTOG 0126 study. IMRT and 3D conformal radiation therapy (3D-CRT) plans were made. Prostate motion and rectum volume changes were simulated in the phantom. IC displacement was corrected by phantom shifting. The delivered dose was measured with IC in 7 cases using intensity-modulated radiation therapy (IMRT) and 3D-CRT fractions, and single square-shaped beams: anteroposterior (AP), posteroanterior (PA), and lateral (LAT). Variations from the calculated doses were slightly below 1% at IMRT and around 1% at 3D-CRT; below 4.5% at square AP beam; up to 9% at square PA beam; and around 0.5% at square LAT beam. Other authors have already shown that by using planning systems and ultrasonic and cone beam CT guidance, correction of organ motions in a real patient during prostate cancer IGRT does not have a significant dosimetric effect. The inspection of our phantom--as described here-ended with similar results. Our team suggested that our model is sufficiently realistic and can be used for IGRT and ART testing.

TIP47 confers resistance to taxol-induced cell death by preventing the nuclear translocation of AIF and Endonuclease G.

Tail-interacting protein (TIP47, also named PP17) has been implicated in lipid droplet metabolism and in the development of late endosomes, to date however, no data about its possible role in regulating cell death processes has been available. Here, we provide evidence for the role of TIP47 in the regulation of mitochondrial membrane stability and cell death. Overexpression of TIP47 protected NIH3T3 cells from taxol-induced cell death, while suppression of TIP47 by siRNA facilitated cell death. TIP47, but not its truncated form, t-TIP47, decreased taxol-induced cell death as determined by propidium iodide and fluorescent Annexin V staining. Recombinant TIP47, but not t-TIP47, partially prevented taxol-induced depolarization of mitochondria in vitro. Overexpression of TIP47, but not its truncated form, prevented the taxol-induced nuclear and cytoplasmic translocation of AIF and Endonuclease G, as well as the taxol-induced depolarization of mitochondria in NIH3T3 cells. Furthermore, overexpression of TIP47 facilitated Bcl-2 expression and suppressed Bax expression in taxol-treated cells. These data show that besides its previously known functions, TIP47 is involved in the regulation of mitochondria-related cell death by directly stabilizing the mitochondrial membrane system and by favorably affecting the expression of Bcl-2 homologues. Since TIP47 is overexpressed in certain tumors, it is possible that TIP47 contributes to the development of cytostatic resistance.

Protective effect of PACAP against doxorubicin-induced cell death in cardiomyocyte culture.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widely distributed endogenous neuropeptide, also occurring in the cardiovascular system. Among others, PACAP has been suggested as a cardioprotective factor. It has been shown that PACAP inhibits cardiac fibrosis and protects cardiomyocytes against oxidative stress and in vitro ischemia/reperfusion. The aim of the present study was to investigate whether PACAP is protective in doxorubicin-induced cell death of cardiomyocytes. Cardiomyocytes were exposed to 1 µM doxorubicin for 24 h, which resulted in a marked reduction of cell viability and a parallel increase of apoptotic cells assessed by MTT test and annexin V/propidium iodide flow cytometry assay. Co-incubation with 20 nM PACAP increased cell viability and reduced the percentage of apoptotic cells. Furthermore, doxorubicin increased the activation of caspase-3 and decreased the phosphorylation of Bad, while simultaneous PACAP treatment reduced the caspase-3 activation and increased the level of phospho-Bad. In summary, our present results demonstrate that PACAP effectively protects cardiomyocytes against doxorubicin-induced apoptotic cell death.

Chemo-radiotherapy in locally advanced squamous cell oesophageal cancer--are upper third tumours more responsive?

Before neoadjuvant therapy was widely applied, the prognosis of oesophageal cancer had been considered dependent on the location of the tumor, i.e. upper third cancers had had the worst prognosis. The aim of this retrolective study was to prove the efficiency of the neoadjuvant treatment, and to compare the response of esophageal cancer in different locations. Between January 1998 and September 2005, 102 patients with locally advanced squamous cell oesophageal cancer received preoperative chemo-radiotherapy. In 40 cases the tumor was located in the upper third and in 62 cases in the middle third of the oesophagus. After a four-week-long treatment free period restaging was carried out and patients considered resectable were submitted to surgery. From 40 patients with upper third oesophageal cancer 28 underwent oesophageal resection or pharyngo-laryngectomy. Thiry-five percent a complete histopathological remission was observed. From 62 patients with middle third oesophageal cancer 43 underwent oesophageal resection. Histological examination of the resected specimens documented complete response only in three patients. The median survival and the R0 resection rate were similar in the two groups. Although the resection rate, perioperative morbidity, mortality and the median survival were similar in the two groups, a significantly higher rate of complete response (p < 0,05) was observed in patients with upper third oesophageal cancer compared to patients with middle third oesophageal cancer. It seems that upper third oesophageal cancer has superior sensitivity to multimodal treatment therefore our results may support that upper third location is not an unfavorable prognostic factor any more.

Facilitation of mitochondrial outer and inner membrane permeabilization and cell death in oxidative stress by a novel Bcl-2 homology 3 domain protein.

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

Potentiation of paclitaxel-induced apoptosis by galectin-13 overexpression via activation of Ask-1-p38-MAP kinase and JNK/SAPK pathways and suppression of Akt and ERK1/2 activation in U-937 human macrophage cells.

Galectin-13 transcripts have been identified in several normal and malignant tissues, but the physiological function of galectin-13 is still poorly understood. Here, we present evidence for its possible role in promoting cell death in the U-937 human macrophage cell line. Transfection of U-937 human macrophages by a galectin-13 cDNA-containing mammalian expression vector increased the galectin-13 level and sensitized the cells to stress stimuli. Galectin-13 overexpression facilitated paclitaxel-induced cell death and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease-G without inducing mitochondrial cytochrome-c release or caspase-3 activation. Immunoblot and immunofluorescence data showed that overexpression of galectin-13 induced long-term activation of c-Jun N-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) pathways, as well as activation of apoptosis signal-regulating kinase-1 (Ask-1) kinase while it suppressed paclitaxel-induced long-term activation of the phosphatidilylositol-3-kinase (PI-3K)-Akt and extracellular signal-regulated kinase (ERK1/2) cytoprotective pathways. In addition, pharmacological inhibition of JNK and p38-MAPK pathways protected the cells from paclitaxel-induced cell death. All this data indicate that galectin-13 overexpression promoted apoptosis presumably by activating the Ask-1 kinase-JNK and p38-MAPK pro-apoptotic pathways and by suppressing the PI-3K-Akt and ERK1/2 cytoprotective pathways.

Preliminary study of TIP47 as a possible new biomarker of cervical dysplasia and invasive carcinoma.

UNLABELLED: The aim of this study was to find a possible clinical use of the tail-interacting protein of 47 kDa (TIP47) and further document its expression in smear cytology, different cervical dysplasias, invasive cervical cancer and metastasis. PATIENTS AND METHODS: A new polyclonal anti-TIP47 antibody was developed and used on smears and histological cervix sections of sixty women with different cytological pathologies. Serum TIP47 level of patients with cervical intraepithelial neoplasia (CIN) or carcinoma in stage IIb, IIIa, and IIIb was monitored during treatment. RESULTS: TIP47 was expressed weakly in the dysplasias, stronger in invasive tumors and in lymph node metastasis. In patients with cervical carcinoma, the serum TIP47 level was found to be elevated; it decreased after therapy and elevated again in relapse. CONCLUSION: According to our results, TIP47 could be a good clinical marker for the early detection in blood of the recurrence of cervical carcinoma.

Critical role of bad phosphorylation by Akt in cytostatic resistance of human bladder cancer cells.

BACKGROUND: Taxol is the most commonly used agent for salvage chemotherapy in transitional cell carcinoma of the urothelium. We examined mechanisms responsible for taxol resistance by using T24 human bladder carcinoma cells. MATERIALS AND METHODS: We used an inhibitor and an activator of the phosphatidylinositol-3 kinase-Akt pathway in cell survival and caspase-3 assays, an HPLC method for determining released cytochrome c and immunoblotting for detecting protein phosphorylation. RESULTS: Activation of Akt increased paclitaxel resistance by increasing Bad phosphorylation, leading to decreased release of mitochondrial cytochrome c and caspase-3-mediated apoptosis. On the other hand, inhibition of Akt prevented paclitaxel resistance by enhancing the effects of paclitaxel on Bad phosphorylation, mitochondrial cytochrome c release and caspase-3-mediated apoptosis, besides diminishing or abolishing the opposing effects of Akt activation. CONCLUSION: Akt-mediated Bad phosphorylation plays an important role in preservation of mitochondrial membrane systems leading to paclitaxel resistance in T24 cells.

[Neoadjuvant radiochemotherapy in the treatment of locally advanced rectal tumors]. / Neoadjuváns radiokemoterápia a lokoregionálisan elorehaladott rectumtumorok ellátásában.

We investigated the response rate and side effects of simultaneous, neoadjuvant radiochemotherapy (RCT) in locally advanced rectal cancer. Between 2005 and 2007, we treated 112 patients in stage II-III rectal carcinoma at the Institute of Oncotherapy, University of Pécs. For staging abdomino-pelvic CT (112) and transrectal US (49) or pelvic MR (10), or PET-CT (1) was performed. Radiation therapy was delivered with 3D CRT-based technique using belly-board with 18 MV photon energy, while patients in prone position. A total dose of 45 Gy (single dose 1.8 Gy) was delivered to the tumor and the pelvic lymph nodes. 5-FU and Ca-folinate was administered concomitantly in the 1st and 5th week of radiotherapy. Four weeks after delivering neoadjuvant RCT the patients' control CT was evaluated according to RECIST criteria. RCT was followed by surgery in 6-9 weeks. We graded the histology using the Mandard regression score system. Side effects were registered using CTCAE v 3.0. Grade 1, 2 or 3 acute gastrointestinal toxicity occurred in 12%, grade 3 hematological toxicity in 9.5% of the patients. The response rate determined by using control CT was 64.85%. According to the Mandard regression score, TRG1 occurred in 15%, TRG2 in 30.4%, TRG3 in 28%, TRG4 in 24% and TRG5 in 2.6% of the cases. Radical surgery was performed in 89 cases, 72 with R0 resection. By assessing the histological samples we found downstaging in 46% of the T and 34.5% of the N stage. We have no information on increased postoperative complications. We followed 86 patients after neoadjuvant therapy. Until March 2009 there was no progression in 48 of our patients. In 13 cases local relapse occurred, and in 25 cases the disease progressed because of distant metastasis, although local control was maintained. 10 patients had local relapse and distant metastases. 17 patients passed away. As a conclusion, neoadjuvant RCT of Stage II-III patients is an effective and well tolerated treatment, allowing for high R0 resection rate and bearing no higher risk for postoperative morbidity.

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors.

BACKGROUND: Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. METHODS: Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. RESULTS: Low grade (grades 1-2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. CONCLUSION: Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.

Preventing apoptotic cell death by a novel small heat shock protein.

NCBI database analysis indicated that the human C1orf41 protein (small heat shock-like protein-Hsp16.2) has sequence similarity with small heat shock proteins (sHsps). Since sHsps have chaperone function, and so prevent aggregation of denatured proteins, we determined whether Hsp16.2 could prevent the heat-induced aggregation of denatured proteins. Under our experimental conditions, recombinant Hsp16.2 prevented aggregation of aldolase and glyceraldehyde-3-phosphate dehydrogenase, and protected Escherichia coli cells from heat stress indicating its chaperone function. Hsp16.2 also formed oligomeric complexes in aqueous solution. Hsp16.2 was found to be expressed at different levels in cell lines and tissues, and was mainly localized to the nucleus and the cytosol, but to a smaller extent, it could be also found in mitochondria. Hsp16.2 could be modified covalently by poly(ADP ribosylation) and acetylation. Hsp16.2 over-expression prevented etoposide-induced cell death as well as the release of mitochondrial cytochrome c and caspase activation. These data suggest that Hsp16.2 can prevent the destabilization of mitochondrial membrane systems and could represent a suitable target for modulating cell death pathways.

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway.

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase-Akt cytoprotective pathway.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL.

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.

Pathological circumstances impair the ability of "dark" neurons to undergo spontaneous recovery.

The effects of dehydrating drugs (furosemide, mannitol and glycerine), potassium channel modulators (tetraethylammonium chloride, 5-hydroxydecanoic acid Na salt, minoxidil and pinacidil), sodium channel modulators (veratridine, brevetoxin-9, 5-(N,N-dimethyl)amiloride and benzamil-HCl) and mitochondrial enzyme inhibitors (3-nitropropionic acid, 2,4-dinitrophenol and chloramphenicol) on the fate of electrically produced "dark" hippocampal dentate granule neurons were investigated. All but one (chloramphenicol) of these bioactive reagents substantially retarded the recovery and increased the death rate of such "dark" neurons. As concerns the dehydrating drugs and ion channel modulators, these effects are considered to be consequences of the fact that relatively large volumes (more than half of the original cell volume) of cytoplasmic fluid (water molecules, inorganic ions and metabolites) leave the affected cells through passive pores within a few minutes. The effects of the mitochondrial enzyme inhibitors appear to indicate that restoration of the original cell volume (recovery) demands metabolic (enzyme-mediated) energy. All these features support our previous assumption that the exogenous circumstances existing acutely after the formation of "dark" neurons in neurological diseases decide whether they will recover or die.

Functional analyses of placental protein 13/galectin-13.

Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity was confirmed by 31P-NMR. In this study, recombinant PP13/galectin-13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin-13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin-13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI-TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin-13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin-like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin-13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin-13 may have special haemostatic and immunobiological functions at the lining of the common feto-maternal blood-spaces or developmental role in the placenta.