Pediatric brain tumors: Update of proteome-based studies.

Pediatric brain tumors (PBTs) are the most common solid malignancies in childhood and continue to pose a serious burden to modern societies. Existing treatments impose debilitating effects on the developing child, highlighting the need for molecularly targeted treatments with reduced toxicity, as well as the necessity of markers that reliably assess efficacy of, and tumor response to targeted-therapies of PBTs. On this regard advances in technologies of protein identification and quantification, the large-scale, high-throughput investigation of the proteome, as well the newly-emerging field of "proteogenomics" aim to further our knowledge towards understanding the molecular pathophysiology of PBTs. This mini review article presents all updates on knowledge produced and published during the last years on PBT research derived from "omics" technologies, mainly involving protein research and proteomics.

Protein expression during early stages of bone regeneration under hydrophobic and hydrophilic titanium domes. A pilot study.

BACKGROUND AND OBJECTIVES: There is significant evidence that, during the early stages of osseointegration, moderately rough hydrophilic (SLActive) surfaces can accelerate osteogenesis and increase bone-to-implant contact in comparison to hydrophobic (SLA) surfaces. However, very little is known regarding the molecular mechanisms behind the influence that surface chemistry modifications to increase hydrophilicity determine on bone healing. The aim of this study was to describe for the first time the proteins and related signalling pathways expressed during early osseous healing stages under SLA and SLActive titanium domes for guided bone regeneration. MATERIAL AND METHODS: One SLA and 1 SLActive dome with an internal diameter of 5.0 mm and a height of 3.0 mm were secured to the parietal bones of nine 6-month-old male New Zealand rabbits. Three animals were randomly euthanized at 4, 7 and 14 days and the newly formed tissues retrieved under the domes were analysed with liquid chromatography-mass spectrometry/mass spectrometry. STRING and KEGG databases were applied for Gene Ontology and pathway analyses. RESULTS: A different modulation of several pathways was detected between the 2 groups at all healing times. The main differences in the osseous healing response associated to the 2 surfaces were related to pathways involved in regulating the inflammatory response, differentiation of osteoblast precursors and skeletogenesis. At day 7, the highest number of proteins and the highest cellular activity were observed in both groups, although a more complex and articulated proteome in terms of cellular metabolism and signal transduction was observed in SLActive samples. CONCLUSION: This is the first study describing the proteome expressed during early healing stages of guided bone regeneration and osseointegration. A combination of enhanced early osteogenic response and reduced inflammatory response were suggested for the hydrophilic group. Future studies are needed to corroborate these findings and explore the molecular effects of different titanium surfaces on the cascade of events taking place during bone formation.

The effect of experimental osteoporosis on bone regeneration: part 2, proteomics results.

OBJECTIVES: To identify and describe protein expression in a Wistar rat calvarial critical size defect (CSD) model following treatment with guided bone regeneration in healthy and osteoporotic conditions. MATERIAL AND METHODS: Thirty-six 10-month-old female Wistar rats were used. Half of them were ovariectomized (OVX) and fed with a low-calcium diet to induce an osteoporotic-like status. In each animal of both groups, two 5-mm calvarial CSDs were treated with deproteinized bovine bone mineral graft particles and a bilayer collagen membrane. Six OVX and six control rats were randomly euthanized at 7, 14, and 30 days. One defect/animal was randomly chosen for proteomic analysis. Differently expressed proteins between the two groups were identified with matrix-assisted laser desorption time-of-flight mass spectrometry and liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: At 7 days, 29 and 27 proteins were, respectively, identified in the healthy and OVX animals. At 14 days, 103 proteins were detected in the healthy controls and 20 proteins in the OVX rats, while at 30 days, 31 and 75 proteins were identified, respectively. Only limited proteins known to play a role in the later stages of bone formation and maturation were identified within the animals 'proteomes. DISCUSSION: The osseous formation process was quite immature even at 30 days of healing. An overexpression of inflammatory and stress response pathways was detected in the OVX animals, as well as a tendency toward a delayed maturation of the osseous wound and a reduced/delayed differentiation of osteoblast cell precursors.

Use of annexin V antibody to identify apoptotic cells during pregnancy.

In a previous study, we demonstrated that apoptosis increased according to gestational age, accounting partly for the presence of free fetal DNA in maternal plasma and serum. Using simultaneous TUNEL assay and FISH analysis, we identified the fetal origin of part of the apoptotic cell population, but very few TUNEL-positive cells showed hybridization signals since they were in a late apoptosis stage and nuclei were destroyed. In the present study, the apoptotic cell population was identified immunocytochemically using Annexin V, a marker of cells in an early stage of apoptosis. The mean apoptosis rate in mononuclear cells isolated from the peripheral blood of 20 pregnant women in the 16th to 19th week of pregnancy with Annexin V was 6.8 +/- 0.5% (range: 4.2-8.1%) compared to 6.14 +/- 0.5% (range: 3.7-6.9%) obtained with ethidium bromide staining. FISH using X and Y chromosome-specific probes was applied in 11 cases known to be carrying male fetuses. Eighty percent of Annexin V+ cells showed hybridization signals, while the proportion of apoptotic cells showing X/Y signals was 7.8% (range: 5-12%). Although our results are still preliminary, it seems that use of Annexin V antibody to detect the apoptotic cell population improves FISH analysis and allows a more accurate estimate of the proportion of fetal cells among the apoptotic cell population.

Fas and Bcl-2 protein expression in thyrocytes of patients with nodular goiter.

OBJECTIVE: The relative expression of the apoptotic protein Fas and the anti-apoptotic protein Bcl-2 were investigated in thyrocytes from patients with non-toxic nodular goiter (NTG, n=20) and Hashimoto's thyroiditis (HT, n=5), who underwent fine-needle aspiration biopsy for diagnostic reasons. On the basis of the clinical and cytological findings, the patients with NTG were sub-classified into the group of those with colloid nodules (n=9), degenerative nodules (n=6) and adenomatous nodules (n=5). METHODS: Fine-needle biopsy aspirates were examined by immunocytochemistry for Fas and Bcl-2 expression, using specific monoclonal antibodies. For the evaluation of Fas and Bcl-2 immuno-reactivity, an expression index, based on the number of cells with positive staining, was used: grade 1 included samples with positive staining in <20% of cells; grade 2 included samples with 20-50% positive cells; and grade 3 included samples with >50% positive cells. RESULTS: Fas protein expression was generally low (grade 1) in patients with nodular goiter, in contrast to patients with HT, in whom high expression was detected (grade 3). Only in aspirates from degenerative nodules (four out of six), and in which lymphocytes were also present, was Fas expressed at an intermediate level (grade 2). On the other hand, Bcl-2 protein was differentially expressed among the nodule subtypes. It was low in colloid and degenerative nodules (grade 1) but high in adenomatous ones (grades 2 and 3). Bcl-2 expression was also low in patients with HT (grade 1). CONCLUSION: It is concluded that in comparison to HT, where there is up-regulation of Fas and down-regulation of Bcl-2 protein, Fas expression is low in human goiter, indicating low apoptotic activity. The regulation of Bcl-2 protein differs between adenomatous and colloid nodules, suggesting that this protein may play a role in the differentiation of thyroid nodules.

Hodgkin's disease in a child with sickle cell disease treated with hydroxyurea.

Hydroxyurea (HU) is an oral drug that ameliorates the clinical course of sickle cell anemia by increasing the levels of fetal hemoglobin and decreasing the adhesion of red cells to endothelium. Although HU has minimal short-term toxicity, few data are available about the long-term safety and the potential risk for carcinogenesis or leukemogenesis. An 8-year-old child with sickle cell/beta 0-thalassemia who received HU treatment for painful crises is described. Six months after the initiation of the HU treatment he developed Hodgkin's disease, lymphocyte predominance subtype. Chemotherapy induced a complete remission. After discontinuation of chemotherapy the painful crises recurred and bone marrow transplantation was decided at the age of 12 years. Two years after the bone marrow transplantation, the child is in complete remission without painful crises. Although the authors suggest that the development of Hodgkin's disease is a coexisting event, questions arise about the safety of HU treatment in childhood.

Apoptosis in maternal peripheral blood during pregnancy.

OBJECTIVE: To investigate the mononuclear cell apoptosis rate during pregnancy. MATERIALS AND METHODS: Apoptosis was quantitated by EtBr staining in whole peripheral blood samples of 135 women in different gestational weeks and 85 nonpregnant women used as controls. Apoptosis was also qualitated by TUNEL assay. RESULTS: The apoptosis rate increased during pregnancy according to gestational age. In chromosomally abnormal fetuses apoptosis was 2.5-fold higher than that found in pregnancies with normal embryos matched for gestational age. FISH in TUNEL-positive cells using X, Y and 21 chromosome probes verified the fetal origin of part of the apoptotic population. CONCLUSION: Apoptosis is stimulated in maternal peripheral blood during pregnancy, possibly accounting partly for the presence of free fetal DNA in maternal serum. The increased apoptosis rate in pregnancies with chromosomally abnormal fetuses may have additional clinical importance.

Low relapse rate in children with acute lymphoblastic leukemia after risk-directed therapy.

PURPOSE: Even though acute lymphoblastic leukemia (ALL) responds well to chemotherapy, relapse remains the major problem. This study documents relapse and survival rates in 85 consecutive children (33 at good risk, 52 at high risk) with ALL diagnosed in 1991 to 1996. PATIENTS AND METHODS: Until 1993, the New York II protocol for the high-risk group and a combination of UKALL XI (induction) and R blocks of ALL-REZ BFM-87 (intensification) regimens for patients at good risk were used. To reduce toxicity, the protocols were subsequently modified. Consolidation treatment was the same for both groups, consisting of a lower cytarabine dose and methotrexate removal, whereas intensification was changed only for the high-risk group using the BB block of the NHL-BFM-90 protocol. The bone marrow clearance of leukemia was assessed on day 22, and minimal residual disease was detected using polymerase chain reaction analysis of Ig heavy-chain gene rearrangements. RESULTS: Seventy patients had common precursor B lineage ALL, six had pre-B-ALL, eight had T-ALL, and one had B-ALL. Two patients never achieved remission and died. Six patients died of consolidation-related complications. Four more patients died, two during induction and two during maintenance therapy. Two other children had relapse (2.3%), both of whom were treated with the earlier protocols and then underwent bone marrow transplantation. Four more children with morphologically complete remission showed minimal residual disease (which reached the levels of 1 leukemic cell among 10(2)-10(4) normal cells) with the use of clone-specific probes at several points of the study intervals, but never had relapse. The 5-year overall and event-free survival rates were 86% and 83%, respectively. The 5-year overall survival rates for good-risk and high-risk groups were 94% and 81%; the corresponding event-free rates were 91% and 78%. The 5-year event-free survival rate in the patients at high risk was significantly higher after the protocol change (90% vs. 65%, P = 0.04). CONCLUSIONS: The modification proved to be effective in diminishing the therapeutic toxicity and improving the efficacy, mainly for the high-risk group.

Metallothionein expression prevents apoptosis. II: Evaluation of the role of metallothionein expression on the chemotherapy-induced apoptosis during the treatment of acute leukemia.

Metallothioneins (MT) are low molecular weight cysteine-rich proteins, present in a wide variety of eukaryotes. Although their physiological function is not entirely understood, recently it was found that in vitro human MTs (hMTs) expression prevents apoptosis. In the present study, the apoptosis preventing effect of hMTs is evaluated in vivo, in order to correlate the apoptotic effect of chemotherapy during the treatment of acute leukemia with the expression of hMTs. The expression of hMTs was studied immunocytochemically in bone marrow smears and peripheral blood cytocentrifugations of 47 children with acute leukemia at diagnosis and during treatment. Apoptosis was quantitatively studied in peripheral blood samples during the induction therapy. Eighteen cases were found to be positive for hMTs expression at diagnosis and the mean apoptosis curve of these cases showed maximal effect on the second day of treatment, the apoptotic action of chemotherapy being completed on the tenth day. The mean apoptosis curve of the hMTs negative cases (29 cases) showed maximal effect on the first day of treatment and the apoptotic action of chemotherapy was completed on the sixth day. When considering the day on which the maximal apoptotic effect appeared and the day on which the apoptotic action of treatment was completed, the results indicated retardation of the chemotherapy-induced apoptosis dependent on hMTs expression, as a result of resistance to treatment. Furthermore, the study of hMTs expression during treatment, showed that although the apoptotic action of chemotherapy eliminates blast cells, a cell population positive for hMTs survived and increased during treatment, since they were able to escape apoptotic cell death. These findings, indicated that in vivo, hMTs constitute a cellular protective mechanism preventing chemotherapy-induced apoptosis, thus regulating the response of patients to treatment.

Study of minimal residual disease in B-cell lineage acute lymphoblastic leukemia of childhood, using clone-specific probes.

In B-cell lineage acute lymphoblastic leukemia (B-ALL), the clonal rearrangements of the immunoglobulin heavy chain gene locus (IgH), can be used as a molecular marker for the detection of minimal residual disease (MRD). Patients in complete remission may still harbor leukemic cells undetectable by conventional methods such as light-microscopic examination, immunophenotyping and cytogenetics. 30 children with B-ALL were screened at diagnosis by polymerase chain reaction (PCR) for their IgH gene repertoire. 7/30 patients were extensively studied using patient-specific oligonucleotide probes derived from the sequence analysis of bone marrow (BM) samples at diagnosis. 210 PCR products from follow-up BM samples corresponding to these 7 patients were hybridized with the appropriate clone-specific probe in order to detect MRD with high sensitivity and specificity. All the patients were in morphological remission during and after therapy. 25/30 patients were PCR positive at diagnosis. 4/7 patients who were examined for MRD had detectable disease in various periods after diagnosis. Molecular signs of residual cells can persist for a long time during and after therapy. Long term follow-up of MRD could determine the period of therapy and predict relapse, indicating therapeutic interventions.

Could cisplatin as a front-line treatment in childhood non-Hodgkin's lymphoma be a promising therapy?

Non-Hodgkin's lymphomas (NHL) were often erroneously diagnosed as other malignancies and treated accordingly. In this study cisplatin combined with vincristine, cyclophosphamide, and Adriamycin was used incidentally as a front-line treatment in seven children with NHL, because the initial histologic diagnosis was that of a sarcoma. After reevaluation three patients had Ki-1 anaplastic large cell lymphoma of T-cell origin, two abdominal B-cell diffuse high-grade NHL, one mediastinal diffuse large B-cell lymphoma, and one B-cell lymphoma in the stomach. They received at least two courses of cisplatin combined regimen and continued with other protocols for NHL. All patients showed an extremely good response from the first course of therapy and the masses vanished completely. They were followed up for a mean time of 29.5 months and are all in complete remission. The data indicate that cisplatin is active against NHL and might be a promising alternative front-line therapy.

Familial medulloblastoma.

Medulloblastomas account for 20% of all primary brain tumors. The vast majority of them are sporadic. Familial medulloblastoma is very rare--only a few cases have been reported worldwide. Most were observed in siblings of the same sex. The affected children presented at various ages and all of them have died, usually within the first 2 years following diagnosis. The authors describe a case of familial medulloblastoma with unusual characteristics: Two siblings of different sex and a second-degree relative have presented at exactly the same age of 18 months. The histologic pattern was the same in all patients, that of desmoplastic medulloblastoma. All patients are alive and remain in remission 12, 5, and 11 years, respectively, after diagnosis. The genetics and the pathogenesis of the disease remain obscure.

Chromosome fragility and predisposition to childhood malignancies.

In order to clarify the possible connection between autosomal folate sensitive Fragile Sites (FS) and genetic susceptibility to haemopoetic disease in children we investigated the frequency and distribution of FS in the Peripheral Blood Lymphocytes (PBL) of 56 children with newly diagnosed and untreated haematologic malignancies and their parents. The incidence was compared with that of 146 normal controls (children and adults). In all patients the Bone Marrow (BM) karyotype was also determined. Heritable FS were detected in 49 patients (87.5%). 20 children had more than one FS and in all cases it was inherited from one of their parents, although there was a significant excess of transmitting mothers. 19 different FS were identified: 14 common, 4 rare and one, 22q11, which has not been previously reported, but it is considered as important as it coincides with the cancer breakpoint resulting in the formation of the Philadelphia (Ph) chromosome. The frequency of FS in the PBL of the patients was significantly higher than in the controls and this increase was independent of any abnormality detected in the malignant cells of the BM. However, patients with an abnormal BM karyotype displayed increased frequency of FS induction as compared to patients with a normal karyotype. In three cases the heritable FS was found to be at or near the breakpoints of the chromosomal rearrangements detected in the malignant cells. The findings are discussed with regard to cancer specific breakpoints, oncogene loci and sites where viral DNA can be inserted to the genome. The results of this study suggest that autosomal folate sensitive FS may increase the risk for haematologic malignancies through a complex mechanism which remains to be clarified.

Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line.

Human metallothioneins (hMTs), are low molecular weight cysteine-rich proteins that constitute the majority of intracellular protein thiols. Their transcription is regulated by metals, glucocorticoids and cytokines, and in certain tissues it is a highly specialized phenomenon. Although their physiological function is not entirely understood, hMTs induction has been observed to be associated with protection from heavy metal toxicity and cellular resistance to cytotoxic anticancer drugs. However, the main problem in the investigation of the physiological function of hMTs is the absence of any known specific inhibitor, as well as the fact that many genes constitute the hMTs family. As the identification of genes preventing apoptosis is of great interest, we attempted to examine the role of hMTs in the apoptotic process by inhibiting their expression in the immature T cell line CCRF-CEM with antisense sequence-specific phosphorothioate oligodeoxynucleotides (ODNs). In the experimental procedure the cells were activated and cultured in medium containing 20% FBS instead of 10%, during maintenance. We found that the inhibition of hMTs synthesis, induced by the incubation of the cells for 24 hours with ODNs, stimulated the apoptotic process, as confirmed by the characteristic morphological alterations and DNA fragmentation. Quantitative analysis of apoptosis has shown that inhibition of hMTs expression results in a dose-dependent and ODNs sequence-specific induction of apoptosis. Immunocyto-chemical detection of hMTs followed by Tunel assay showed that all the Tunel positive cells were hMTs negative, suggesting that hMTs expression prevents apoptosis. As hMTs induction is rapid and transient in response to stress and/or environmental stimuli, these results indicate that hMTs constitute a cellular protective mechanism, neutralizing external apoptotic signals.

Cadmium induces apoptosis differentially on immune system cell lines.

We investigate the role of cadmium-induced apoptosis in the immune system, studying the apoptotic effect of Cd2+ in three human cell lines, the T-cell line CCRF-CEM, the B-cell line Raji and the lymphoblastoid cell line Molt-3. Cd2+ was found to be dose-dependently toxic for these cell lines, after 18 h incubation. The 50% lethal dose (LD50) for CCRF-CEM was 25 +/- 20 microM, for Molt-3 was 22.5 +/- 2.4 microM, and for Raji was 13.5 +/- 2.2 microM. DNA electrophoresis and quantitation of apoptosis after 18 h incubation with different Cd2+ concentrations was carried out. In CCRF-CEM cells, apoptosis was detected at 10 microM, reaching a maximum at 30 microM. In Molt-3, apoptosis was detected at 10 microM, increased thereafter and a plateau effect was observed from 30 to 50 microM Cd2+. In Raji, apoptosis was detected at 5 microM, while a plateau effect was observed from 20 to 30 microM Cd2+. The above results indicated that Raji cells were more sensitive to cadmium compared to both CCRF-CEM and Molt-3 cells, suggesting a differential Cd2+-induced apoptotic effect, which may disturb the immune system normal growth and development.

Fetal cells in maternal blood: isolation by magnetic cell sorting and confirmation by immunophenotyping and FISH.

Fetal cells entering the maternal circulation during pregnancy constitute a potential source for safe and reliable non invasive prenatal diagnosis. However, selecting the appropriate fetal cell type and methods of enrichment are areas of paramount importance. Most investigators consider fetal nucleated red blood cells (NRBCs) to be the cell type of choice, since they are mononuclear, abundant in fetal blood, relatively well differentiated and have a limited life span. Twenty ml of peripheral blood samples were collected from 40 pregnant women in the 16th to 18th week of pregnancy. To enrich for NRBCs, found within an excess of maternal cells, negative magnetic cell sorting (MACS) was used. Leukocytes were depleted from maternal blood by treatment with anti CD45 monoclonal antibody, as this surface antigen is not expressed in NRBCs. NRBCs were detected in 35 of the 40 maternal samples with May Grunwald-Giemsa staining. In 30 cases UCH gamma positive cells were identified after immunophenotyping with a monoclonal antibody directed against the gamma chain of fetal hemoglobin. The mean number of isolated NRBCs was 6 (range 1-15). In 5 cases we were able to successfully perform FISH on the immunophenotyped cells and determine correctly the fetal gender using X and Y chromosome specific probes.

Study of apoptosis in peripheral blood of patients with acute lymphoblastic leukemia during induction therapy.

In acute lymphoblastic leukemia (ALL) the apoptosis of blast cells in peripheral blood (PB) and bone marrow before and/or during treatment, is of great interest. As the morphological changes during apoptosis provide the most reliable markers, in the present study we utilized a nuclear stain based on ethidium bromide (EtBr) for the rapid qualitative and quantitative measurement of circulating apoptotic cells directly in PB suspensions without fractionation. By using a fluorescent microscope the apoptotic cells appeared clearly visible, making the estimation of their percentage straightforward. We studied apoptosis before and during the onset of chemotherapy in PB from 16 children with ALL at diagnosis, and one upon relapse. In the cases studied at diagnosis the circulating apoptotic cells were found in variable percentages after 24 hours of treatment. Maximal apoptosis was observed after 24 hours of treatment in five cases and after 48 hours in two cases. After 96 hours of treatment the cases studied at diagnosis could be divided into three groups: those with a) negligible apoptotic cells, b) between 8% and 12% apoptotic cells and c) a high percentage of apoptotic cells (more than 20%). The relapsed case was characterized by P-glycoprotein positive blast cells, and circulating apoptotic cells which remained very low at all time points. Thus, it is possible to evaluate the response to treatment by studying apoptosis directly in peripheral blood. Therefore, the maximum apoptotic effect and the percentage of circulating apoptotic cells at the different time intervals must be considered.

Development of a quantitative method for the study of apoptosis in peripheral blood.

In vitro and ex vivo many methods are effective for the quantitation of apoptotic cells. These methods cannot be applied for the study of apoptosis in peripheral blood (PB) samples, because of the disappearance of circulating apoptotic cells and the heterogeneity of cell populations. In the present study we describe a nuclear chromatin staining method using ethidium bromide (EtBr), by which we were able to identify and quantify morphologically apoptotic cells in an early stage of the apoptotic process, before their disappearance. The application of the EtBr nuclear stain in CCRF-CEM cells incubated with epirubicin, provides a reliable and reproducible estimation of apoptosis compared with other assays. The main advantage of this method is that it permits the study of four cellular subpopulations by their morphological characteristics (normal cells, early apoptotic, late apoptotic and cells which died from necrosis). Furthermore, we utilized the EtBr nuclear stain to identify and quantify the circulating apoptotic cells directly in the PB of patients with acute lymphoblastic leukemia during multidrug chemotherapy. Circulating apoptotic cells were detectable after 24 hours treatment followed by the decrease of peripheral blood mononuclear cells. Thus staining of nuclei with EtBr provides a rapid and useful method for the quantitative study of apoptotic cells in somatic fluids during clinical trials.

Modulation of stress proteins by Cd2+ in a human T cell line.

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.