Brazilian Pampa Biome Honey Protects Against Mortality, Locomotor Deficits and Oxidative Stress Induced by Hypoxia/Reperfusion in Adult Drosophila melanogaster.

We aimed to investigate the potential beneficial effects of the Brazilian Pampa biome honey in a Drosophila-based hypoxia model. Adult flies were reared in standard medium in the presence or absence of honey (at a final concentration of 10 % in medium). Then, control flies (4 % sucrose in medium) and honey-treated flies were submitted to hypoxia. Subsequently, flies were analyzed for mortality, neurolocomotor behavior (negative geotaxis), mitochondrial/oxidative stress parameters and expression of hypoxia/stress related genes by RT-qPCR. The HPLC analysis revealed the presence of phenolics and flavonoids in the studied honey. Caffeic acid was the major compound followed by p-coumaric acid and kaempferol. The presence of such compounds was correlated with a substantial antioxidant activity in vitro. Flies subjected to hypoxia presented marked mortality, locomotor deficits and changes in oxidative stress and mitochondrial activity parameters. Honey treatment was able to completely block mortality and locomotor phenotypes. In addition, honey was able to reverse ROS production and hypoxia-induced changes in mitochondrial complex I and II activity. Hypoxia also induced an up-regulation in mRNA expression of Sima (HIF-1), NFκß, NRF2, HOX, AKT-1, InR, dILP2, dILP5 and HSP27. Honey treatment was not able to modulate changes in the tested genes, indicating that its protective effects involve additional mechanisms other than transcriptional activity of hypoxia-driven adaptive responses in flies. Our results demonstrated, for the first time, the beneficial effects of honey against the deleterious effects of hypoxia/reperfusion processes in a complex organism.

Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC-driven neuroblastoma: a Children's Oncology Group study.

BACKGROUND: MYCN amplification with subsequent MYCN protein overexpression is a powerful indicator of poor prognosis of neuroblastoma patients. Little is known regarding the prognostic significance of the homologous MYC protein expression in neuroblastoma. METHODS: Immunostaining for MYCN and MYC protein was performed on 357 undifferentiated/poorly differentiated neuroblastomas. Results were analysed with other prognostic markers. RESULTS: Sixty-seven (19%) tumours were MYCN(+), 38 (11%) were MYC(+), and one(0.3%) had both proteins(+). MYCN(+) tumours and MYC(+) tumours were more likely diagnosed in children>18months with stage4-disease. MYCN(+) tumours were associated with amplified MYCN, Unfavourable Histology (UH), and High-MKI (Mitosis-Karyorrhexis Index). MYC(+) tumours were also frequently UH but not associated with MYCN amplification, and more likely to have low-/intermediate-MKI. Favourable Histology patients without MYC/MYCN expressions exhibited the best survival (N=167, 89.7±5.5% 3-year EFS, 97.0±3.2% 3-year OS), followed by UH patients without MYC/MYCN expressions (N=84, 63.1±13.6% 3-year EFS, 83.5±9.4% 3-year OS). MYCN(+)patients and MYC(+)patients had similar and significantly low (P<0.0001) survivals (46.2±12.0% 3-year EFS, 63.2±12.1% 3-year OS and 43.4±23.1% 3-year EFS, 63.5±19.2% 3-year OS, respectively). Notably, the prognostic impact imparted by MYC expression was independent from other markers. CONCLUSIONS: In this series, ∼30% of neuroblastomas had augmented MYCN or MYC expression with dismal survivals. Prospective study of MYC/MYCN protein expression signature as a new biomarker for high-risk neuroblastomas should be conducted.

Synergistic interaction of macrophages and lymphocytes in antigen-induced transformation of lymphocytes.

The role of macrophages and lymphocytes in antigen-induced transformation of lymphocytes has been investigated. Lymphocytes and macrophages were obtained from inbred strain 13 guinea pigs which were either unimmunized or immunized with complete Freund's adjuvant (CFA) or tetanus toxoid in CFA. The transformation response to PPD or tetanus toxoid was assayed by tritiated thymidine incorporation. Addition of macrophages to immune lymphocytes significantly increased their response to purified protein derivative (PPD) or tetanus toxoid. This was observed if the macrophages were (a) "immune" or "nonimmune", (b) unirradiated or irradiated (3000 R), (c) 99% pure, and (d) peritoneal or alveolar in origin. Neither immune nor nonimmune macrophages were able to induce nonimmune lymphocytes to respond to PPD or tetanus toxoid. When macrophages were incubated with PPD or tetanus toxoid and then washed, they stimulated immune lymphocytes to transform. An incubation time of (1/2) hr was adequate, however, 2-4: hr was optimal. These studies indicate (a) that antigen-induced transformation of lymphocytes is greatly enhanced by macrophages; (b) that macrophage-antigen interaction can antecede lymphocyte-antigen interaction and results in macrophages which are able to stimulate lymphocyte transformation; and (c) that the immunological memory requisite to elicit specific transformation responses is a property of the lymphocyte and not the macrophage.

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside G(D2) and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups.

Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage.

A domain of DNA designated N-myc is amplified 20- to 140-fold in human neuroblastoma cell lines but not in cell lines from other tumor types. N-myc has now been found to be amplified in neuroblastoma tissue from 24 of 63 untreated patients (38 percent). The extent of amplification appears to be bimodal, with amplification of 100- to 300-fold in 12 cases and 3- to 10-fold in 10 others. Amplification was found in 0 of 15 patients with stage 1 or 2 disease, whereas 24 of 48 cases (50 percent) with stage 3 or 4 had evidence of N-myc amplification. These data indicate that N-myc amplification is a common event in untreated human neuroblastomas. Furthermore, N-myc amplification is highly correlated with advanced stages of disease (P less than 0.001) and with the ability to grow in vitro as an established cell line, both of which are associated with a poor prognosis.

Identification and characterization of the protein encoded by the human N-myc oncogene.

The human N-myc gene is related to the c-myc proto-oncogene, and has been shown to have transforming potential in vitro. Many studies have reported amplification of N-myc in human neuroblastoma and retinoblastoma cell lines. In primary tumors, amplification of the gene was found to correlate directly with behavior of the tumor. Specific restriction fragments of a partial complementary DNA clone of N-myc from LA-N-5 human neuroblastoma cells were placed into a bacterial expression vector for the purpose of producing antigens representative of the N-myc protein. Rabbits immunized with these antigens produced antisera that recognized a protein of 62-64 kilodaltons in neuroblastoma cells. By several criteria, this protein appears to be part of the same proto-oncogene family as the c-myc protein. Moreover, the antisera to fragments of this protein were capable of histochemically identifying malignant cells in clinical specimens.

Observation of magnetic skyrmions in unpatterned symmetric multilayers at room temperature and zero magnetic field.

Magnetic skyrmions are promising candidates for the next generation of spintronic devices due to their small size and topologically protected structure. One challenge for using these magnetic states in applications lies on controlling the nucleation process and stabilization that usually requires an external force. Here, we report on the evidence of skyrmions in unpatterned symmetric Pd/Co/Pd multilayers at room temperature without prior application of neither electric current nor magnetic field. Decreasing the ferromagnetic interlayer thickness, the tuning of the physical properties across the ferromagnetic/non-magnetic interface gives rise to a transition from worm like domains patterns to isolated skyrmions as demonstrated by magnetic force microscopy. On the direct comparison of the measured and simulated skyrmions size, the interfacial Dzyaloshinskii-Moriya interaction (iDMI) was estimated, reveling that isolated skyrmions are just stabilized at zero magnetic field taking into account non-null values of iDMI. Our findings provide new insights towards the use of stabilized skyrmions for room temperature devices in nominally symmetric multilayers.

Volumetric and functional heterogeneity of human monocytes.

Volume analysis of purified human blood monocytes revealed distinct populations of large and small cells. Computer curve fitting suggested a third, intermediate-sized population. These monocytes were designated M1, M2, and M3 in order of increasing size, and their approximate volumes were 150, 250, and 480 micron3, respectively. The three subpopulations were present in all 30 normal individuals tested. Two new techniques were developed that separate monocytes into M1 + M2 and M3 fractions; one used preferential incorporation of carbonyl iron particles by M3 cells and the other used the selective aggregation of M3 cells by thrombin in the presence of platelets. The chemotactic response to zymosan-activated human serum by total monocytes, M1 + M2 monocytes, and M3 monocytes was determined by the agarose plate method. In all experiments M3 monocytes were 10-fold more responsive than M1 + M2 monocytes and were significantly more so than total monocytes. These findings suggest that M3 cells are the major subpopulation capable of directional migration. This investigation establishes the existence of volumetrically definable subpopulations of human monocytes that are functionally distinct.

Chromogranin A in children with neuroblastoma. Serum concentration parallels disease stage and predicts survival.

Chromogranin A is an acidic protein costored and coreleased with catecholamines from storage vesicles. Its serum concentration is elevated in patients with peptide-producing endocrine neoplasia. We measured serum chromogranin A at the time of diagnosis in 34 children with all stages of neuroblastoma. With a sensitivity of 91% and specificity of 100%, serum chromogranin A emerged as a useful diagnostic tool for neuroblastoma, comparable to or better than other measurements such as neuron-specific enolase, ferritin, or dopamine-beta-hydroxylase. Mean serum chromogranin A correlated with disease stage (r = 0.76, P less than 0.01). The relationship of prognosis (progression-free survival) to baseline serum chromogranin A, age, and disease stage was determined in 34 patients at risk for relapse, with a median followup period of 18 mo (range, 1-48 mo). The survival rate for patients with lower serum chromogranin A levels (less than 190 ng/ml at the time of diagnosis) was 69%, whereas it was 30% for those with higher chromogranin A levels (P less than 0.05). Furthermore, when subjects were additionally stratified by either age or stage, chromogranin A was an effective prognostic tool in patients who either were older than 1 yr (P less than 0.005) or had more advanced disease (stage III or IV; P less than 0.05). We conclude that serum chromogranin A in neuroblastoma is (a) a valuable (sensitive and specific) diagnostic tool, (b) a correlate of tumor burden, and (c) a useful predictor of survival.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Characterization of N-myc amplification units in human neuroblastoma cells.

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.

Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children's Oncology Group.

PURPOSE: In the Children's Oncology Group, risk group assignment for neuroblastoma is critical for therapeutic decisions, and patients are stratified by International Neuroblastoma Staging System stage, MYCN status, ploidy, Shimada histopathology, and diagnosis age. Age less than 365 days has been associated with favorable outcome, but recent studies suggest that older age cutoff may improve prognostic precision. METHODS: To identify the optimal age cutoff, we retrospectively analyzed data from the Pediatric Oncology Group biology study 9047 and Children's Cancer Group studies 321p1-p4, 3881, 3891, and B973 on 3,666 patients (1986 to 2001) with documented ages and follow-up data. Twenty-seven separate analyses, one for each different age cutoff (adjusting for MYCN and stage), tested age influence on outcome. The cutoff that maximized outcome difference between younger and older patients was selected. RESULTS: Thirty-seven percent of patients were younger than 365 days, and 64% were > or = 365 days old (4-year event-free survival [EFS] rate +/- SE: 83% +/- 1% [n = 1,339] and 45% +/- 1% [n = 2,327], respectively; P < .0001). Graphical analyses revealed the continuous nature of the prognostic contribution of age to outcome. The optimal 460-day cutoff we selected maximized the outcome difference between younger and older patients. Forty-three percent were younger than 460 days, and 57% were > or = 460 days old (4-year EFS rate +/- SE: 82% +/- 1% [n = 1,589] and 42% +/- 1% [n = 2,077], respectively; P < .0001). Using a 460-day cutoff (assuming stage 4, MYCN-amplified patients remain high-risk), 5% of patients (365 to 460 days: 4-year EFS 92% +/- 3%; n = 135) fell into a lower risk group. CONCLUSION: The prognostic contribution of age to outcome is continuous in nature. Within clinically relevant risk stratification, statistical support exists for an age cutoff of 460 days.

Lack of high-affinity nerve growth factor receptors in aggressive neuroblastomas.

BACKGROUND: Neuroblastoma is a malignancy of the sympathetic nervous system. Nerve growth factor, which has a major role in development of the sympathetic nervous system, has high-affinity (gp140TRK-A) and low-affinity (gp75NGFR) cell-surface receptors. We recently reported preliminary study results showing a lack of gp140TRK-A receptors and rapid disease progression in neuroblastomas, particularly those with amplification of the N-myc (also known as MYCN) proto-oncogene. PURPOSE: This retrospective study was designed to determine if expression of nerve growth factor receptor messenger RNA (mRNA) was associated with biologic and clinical parameters and with survival in neuroblastoma. METHODS: We obtained 80 untreated primary neuroblastomas that had been snap-frozen and stored after surgical excision. To determine expression of gp140TRK-A and gp75NGFR, we performed Northern blot analyses on total RNA from the specimens. Samples from the same specimens were examined for N-myc proto-oncogene amplification, RNA expression, and histologic differentiation, and clinical stage at diagnosis and survival were determined. RESULTS: Of the 80 neuroblastomas, 65 (81%) expressed gp140TRK-A RNA. However, three (27%) of the 11 tumors with genomic amplification and high expression of N-myc RNA and 62 (90%) of the 69 without genomic amplification or detectable N-myc RNA expressed gp140TRK-A mRNA. The inverse relationship between gp140TRK-A mRNA and N-myc expression had high statistical significance (P < .0001). Of the 67 tumors assessable for histologic differentiation, the 13 lacking gp140TRK-A mRNA were histologically undifferentiated, whereas 19 (35%) of the 54 expressing it were differentiated (P = .041). Only 10 (53%) of the 19 metastatic (stage IV) tumors expressed gp140TRK-A mRNA, compared with 90% for other stages (P = .0003). Survival 2 years after diagnosis was 92%, 78%, and 14% for patients whose tumors expressed high, intermediate, and no gp140TRK-A mRNA, respectively (P < .0001). Univariate and multivariate analyses demonstrated that N-myc and gp140TRK-A expression of mRNA and clinical staging were independent predictors of survival. Expression of gp75NGFR mRNA did not correlate with gp140TRK-A mRNA expression, histologic differentiation, stage, or survival. CONCLUSIONS: The expression of gp140TRK-A mRNA correlates with distinct biologic and clinical subsets of neuroblastoma, which suggests a role for the high-affinity nerve growth factor receptors in determining the phenotype of neuroblastoma. The absence of gp140TRK-A mRNA expression, whether or not the N-myc proto-oncogene is amplified, is associated with tumor progression.

Analysis of human tumor cells for Ia-like antigens with monoclonal antibodies.

Cultured and noncultured human solid tumors were analyzed for expression of Ia-like antigens with the use of two monoclonal antibodies and a rabbit antiserum against human Ia-like antigens. Of 27 tumor cells tested, 3 melanomas bound antibodies [e.g., 21,563 cpm 131I-labeled staphylococcal protein A ([131I]SpA) with monoclonal antibody Q5/6], but 24 others (1 melanoma, 9 neuroblastomas, 1 medulloblastoma, 3 gliomas, 4 sarcomas, 2 colon carcinomas, 2 transitional cell carcinomas of the bladder, 1 teratoma, and 1 squamous cell carcinoma of the lung) did so minimally or not at all (0-427 cpm [131I]SpA with antibody Q5/6. Monoclonal antibody Q5/6 was quantitatively absorbed with homogenates of 32 noncultured tumors to determine if Ia-like antigens were expressed by neoplastic cells in vivo. Ten milligrams (wet wt) each of 5 of 7 noncultured melanomas removed more than 83% (median, 85%) of the antibody. In contrast, 10 mg each of 10 neuroblastomas, 7 carcinomas, 4 sarcomas, and 4 Wilms' tumors removed less than 47% (median, 19%) of the antibody; even 100 mg of these tumors removed less than 68% (median, 44%) of the antibody.

Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)- retinamide in neuroblastoma cell lines.

BACKGROUND: The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines, probably in part due to its ability to increase levels of reactive oxygen species (ROS). We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tissue compartment, and many chemotherapeutic agents are antagonized by hypoxia, our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of physiologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death. METHODS: ROS generation was measured by carboxydichlorofluorescein diacetate fluoresence. Ceramide was quantified by radiolabeling and thin-layer chromatography. Immunoblotting was used to assess p53 protein levels. Apoptosis (programmed cell death) and necrosis were analyzed by nuclear morphology and internucleosomal DNA fragmentation patterns. Cytotoxicity was measured by a fluorescence-based assay employing digital imaging microscopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-fmk. Statistical tests were two-sided. RESULTS/CONCLUSIONS: In addition to increasing ROS, 4-HPR (2.5-10 microM) statistically significantly increased the level of intracellular ceramide (up to approximately 10-fold; P<.001) in a dose-dependent manner in two neuroblastoma cell lines, one of which is highly resistant to alkylating agents and to etoposide. Cell death induced by 4-HPR was reduced but not abrogated by hypoxia in the presence or absence of an antioxidant, N-acetyl-L-cysteine. Expression of p53 protein was not affected by 4-HPR. Furthermore, the pan-caspase enzyme inhibitor BOC-d-fmk prevented apoptosis, but not necrosis, and only partially decreased cytotoxicity induced by 4-HPR, indicating that 4-HPR induced both apoptosis and necrosis in neuroblastoma cells. IMPLICATIONS: 4-HPR may form the basis for a novel, p53-independent chemotherapy that operates through increased intracellular levels of ceramide and that retains cytotoxicity under reduced oxygen conditions.

Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis.

BACKGROUND: Neuroblastomas show different histopathologic phenotypes, and the tumor cells can carry normal or multiple copies of the N-myc proto-oncogene (MYCN). Studies of the N-myc gene and histopathology of untreated primary neuroblastomas have demonstrated that both these factors are important in risk assessment. PURPOSE: Our purpose was to determine if there are any associations between N-myc gene copy number, histopathologic features, clinical stage, and progression-free survival (PFS) and if joint analyses of histopathology and N-myc gene copy number improve risk assessment. METHODS: The histopathologic phenotype and N-myc gene copy number were determined for 232 biopsy/surgery specimens obtained from untreated primary neuroblastoma patients. Tumors were classified as having favorable or unfavorable histology on the basis of Schwannian stroma (rich versus poor), neuroblastic differentiation (differentiating versus undifferentiated), and mitosis-karyorrhexis (fragmenting nucleus) index (MKI; high, intermediate, or low) in the context of age at diagnosis (Shimada classification). N-myc gene amplification was considered significant when the gene copy number was at least 10-fold higher than normal as determined by Southern blot analysis. Otherwise, tumors were classified as nonamplified for N-myc. RESULTS: Among 19 stroma-rich tumors, 11 had grossly visible neuroblastic nodules, and two of these had N-myc amplification. Of 213 stroma-poor tumors, 51 had N-myc amplification, all of which were undifferentiated, and 45 (88% of 51) had high MKI. This histologic phenotype was present in less than 10% of tumors with nonamplified N-myc. Of 162 stroma-poor tumors that showed nonamplified N-myc, 45 (28%) were differentiating and 121 (75%) had low MKI. Neuroblastomas of clinical stages I, II, and IV-S nearly always had favorable histology and no amplification of N-myc. Stage III (regional) and particularly stage IV (metastatic) tumors, however, frequently had unfavorable histologic features with or without N-myc amplification. The estimated PFS at the end of 4 years after diagnosis was 83% for patients whose tumors had favorable histology and no N-myc amplification. The estimated PFS for the patients whose neuroblastomas had unfavorable histology, however, was 29% without and 13% with N-myc amplification, respectively. Subsets of patients with stage II, III, or IV disease were identified by both histologic evaluation and N-myc analysis. Multivariate Cox regression analysis indicated that both the histologic and N-myc-based stratifications provided prognostic information that was independent of staging. CONCLUSIONS: Neuroblastomas with N-myc amplification have a characteristic histopathologic phenotype and an aggressive clinical course. In contrast, neuroblastomas without N-myc amplification exhibit a wide range of histologic features that can define prognostic subsets.

Hsp27 expression in neuroblastoma: correlation with disease stage.

BACKGROUND: The 27-kd heat shock protein (Hsp27) is differentially expressed in some malignancies, including breast carcinoma, leukemia, and malignant fibrous histiocytoma. In breast carcinoma, a high-level expression of Hsp27 has been associated with shorter disease-free survival in patients with localized disease. PURPOSE: We have observed variable levels of Hsp27 among neuroblastoma tumors. Our aim in this study was to investigate the relationship between Hsp27 expression and stage of the disease and N-myc gene copy number. METHODS: We determined Hsp27 protein levels in 53 neuroblastoma tumors representing different stages of the disease and in 17 neuroblastoma cell lines by quantitative two-dimensional polyacrylamide gel electrophoresis (PAGE). We also performed statistical analysis of Hsp27 levels in relation to stage of the disease and to N-myc gene copy number. RESULTS: Increased Hsp27 expression in neuroblastomas was associated with limited stage disease and inversely correlated with N-myc gene amplification, a feature known to predict poor clinical outcome. An inverse correlation was also observed between N-myc gene amplification and Hsp27 protein levels among the neuroblastoma cell lines analyzed. Immunohistochemical staining of sections of neuroblastomas showed that Hsp27 was most prominently expressed in the cytoplasm of large ganglionic tumor cells present in neuronally differentiated areas of the tumors. Induction of neuronal differentiation in SMS-KCNR neuroblastoma cells using retinoic acid resulted in an increase in Hsp27. CONCLUSION: High level expression of Hsp27 in neuroblastoma is a feature of limited stage, differentiated tumors. IMPLICATION: Hsp27 may play a part in the biology of neuroblastomas with a favorable outcome.

Disease and non-disease-related cell-mediated cytotoxicity in humans.

The human bladder cancer/T24 system was used to investigate disease and non-disease-related cell-mediated cytotoxicity (CMC). CMC was determined in a modification of the microcytotoxicity assay of Takasugi and Klein. Analysis of data of groups of patients confirmed previous findings that effector cells (EC) from bladder cancer patients were more cytotoxic against T24 than were EC from normal individuals or from patients with other genitourinary cancers. Differences between patients with bladder cancer and other patients were not observed for other target cells. During the course of these experiments, non-disease-associated CMC by EC from individual normal donors and patients was observed. This phenomenon was investigated to determine its reproducibility and its relationship to different methods of preparing EC. Reproducibility of non-disease-related CMC was ascertained using EC prepared from heparinized blood by centrifugation over Ficoll-Hypaque (FH). A total of 126 experiments were performed in which 18 normal donors were tested 2 to 7 times each against 4 target cell lines. Of the resulting 46 combinations or groups of repeated assays, only 7 showed significant variability. Each normal donor had consistent CMC with differences from others being reproducible. CMC was therefore not due to crowding or physical effects. CMC mediated by EC prepared in this manner was then compared to that mediated by EC prepared by other methods in simultaneous tests.

Expression of GD2 ganglioside by untreated primary human neuroblastomas.

Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the frequency of tumors expressing a specific glycosphingolipid, GD2 ganglioside. Total tissue gangliosides were purified by a new partition method, quantitated, and analyzed by high-performance thin-layer chromatography. All 36 neuroblastoma tumors, representing all clinical stages, contained GD2 ganglioside. The mean relative and absolute concentrations of GD2 were substantial (12% of the total tissue gangliosides and 50 nmol/g of tissue) and were independent of the clinical stage of the tumor. In contrast, 6 samples of related but more differentiated tumors (ganglioneuroblastoma and ganglioneuroma) had little or no detectable GD2 (less than or equal to 1.5% of total gangliosides and less than or equal to 4 nmol/g of tissue). These results suggest that GD2 is a sensitive marker for neuroblastoma tissue and may be an excellent target antigen for immunotherapy of this tumor.

Maintenance of normal imprinting of H19 and IGF2 genes in neuroblastoma.

H19 and insulin-like growth factor II (IGF2) are among a few genes which have been confirmed to be imprinted in normal human embryonal tissues. This results in monoallelic expression of maternal H19 and paternal IGF2. Loss of imprinting of these genes producing biallelic expression has been observed in Wilm's tumor and embryonal rhabdomyosarcoma, suggesting that an epigenetic change of DNA, in addition to a genetic change in oncogene(s) and/or tumor suppressor gene(s), may be involved in the development of these childhood cancers. Neuroblastoma, which is an embryonal tumor originating from neural crest-derived cells, occasionally occurs in individuals with the Beckwith-Wiedemann syndrome; Wilm's tumor and embryonal rhabdomyosarcoma occur even more frequently in the Beckwith-Wiedemann syndrome; and paternal uniparental disomy of H19 and IGF2 loci (chromosome 11p15) is present in the Beckwith-Wiedemann syndrome. Furthermore, neuroblastoma cell lines express IGF2, and autocrine/paracrine effects of IGF2 have been demonstrated in these cells. Thus, we examined for imprinting of both H19 and IGF2 in primary untreated neuroblastomas using the RsaI and ApaI polymorphisms within these genes, respectively. Seven of 15 tumors were informative for H19 and for IGF2, and all of these cases showed monoallelic expression of both of these genes. These results indicate that loss of imprinting of H19 and IGF2 does not occur in neuroblastomas.