Persistent alpha 1 integrin subunit expression in human neuroblastoma cell lines which overexpress N-myc and downregulate other integrin subunits

Neuroblastoma is characterized by amplification and overexpression of N-myc. N-myc down-regulates expression of the beta 1 integrin extracellular matrix receptor, however, some beta 1 was found on the surface of N-myc overexpressing neuroblastoma cell lines. It was determined that the alpha 1 subunit is expressed in the cells, associates with beta 1 and is present on the cell surface. Interestingly, the level of alpha 1 was reduced marginally when compared to beta 1. Finally, overexpression of N-myc alters cell morphology on extracellular matrix proteins with persistence of alpha 1 beta 1-dependent responses. Our work supports the conclusion that altered integrin expression may be an important factor in human neuroblastoma.     

Differentiation-related expression of adhesion molecules and receptors on human neuroblastoma tissues, cell lines and variants.

The expression of cellular adhesion molecules (CAM) involved in cell adhesion and immune recognition was measured on neuroblastoma tissue samples, on a neuroblastoma (NB) cell line, SK-N-SH, and on 3 phenotypically different variants, SH-SY5Y, SH-EP, SH-IN, representing neuronal, Schwannian/glial or intermediate NB-cell types. Immunohistochemical analysis of CAM expression by NB and related tumors at different stages of differentiation revealed a co-expression of several CAM (ICAM-1/CD54, LFA-3, VLA-2 and HLA-ABC) associated with low stages and more highly differentiated NB tumors and peripheral neuroepitheliomas (PN). In contrast, N-CAM was uniformly expressed on all NB tumors. Flow cytometric analysis of CAM surface expression by SK-N-SH and variant cells revealed highly variable phenotypes. Expression of ICAM-1, LFA-3, VLA-2 and HLA-ABC molecules was associated with the epithelial cell type represented by the SH-EP variant. In contrast, low expression of these molecules and high expression of N-CAM was associated with the neuronal SH-SY5Y cells. Exposure of the NB cells to differentiation inducers (retinoic acid, 5'-bromodeoxyuridine and phorbol esters) and cytokines (tau-interferon, alpha-tumor necrosis factor) resulted in a variable up-regulation of the expression of all CAMs, except N-CAM, regardless of the type of differentiation induced. In an attempt to establish a link between the pattern of expression of CAM on NB cells and their susceptibility to natural killer (NK) or lymphokine-activated killer (LAK) cell lysis, the analysis revealed that NB cells expressing CAM and a differentiated phenotype were less susceptible to NK lysis, but no difference in the sensitivity of the NB cell types to LAK effectors was observed. Treatment of NB target cells with cytokines or PMA decreased their susceptibility to NK and LAK lysis, while induction of differentiation with RA or BUdR resulted in no changes in the sensitivity to NK and LAK lysis. In conclusion, expression of HLA-ABC and several co-regulated CAMs was shown to be associated with a differentiated phenotype in NB, with an overall decreased sensitivity to NK/LAK effector cells.     

Low complex ganglioside expression characterizes human neuroblastoma cell lines

Low (< or = 35%) or absent expression of the complex 'b' pathway gangliosides GD1b, GT1b and GQ1b (CbG) correlates with an aggressive biological phenotype in human neuroblastoma tumors. To develop an in vitro model to probe mechanisms by which CbG may contribute to neuroblastoma behavior, we have comprehensively evaluated ganglioside expression in nine well-established human neuroblastoma cell lines, all derived from poor prognosis tumors. Total cellular ganglioside content ranged from 8 to 69 nmol/10(8) cells. High performance thin layer chromatography revealed that the simple disialoganglioside GD2 was prominent in eight of the cell lines (up to 60% of total gangliosides), whereas CbG were low (1-21%) in all nine cell lines. The structurally most complex 'b' pathway species, GQ1b, was not detected in any of the cell lines. The prominence of GD2 in neuroblastoma cell lines mirrors the high expression of GD2 that characterizes human neuroblastoma tumors, and the low CbG expression in the cell lines is analogous to that found in clinically and biologically unfavorable neuroblastoma tumors, thus establishing these neuroblastoma cell lines as valuable model systems for study of the role of CbG in the pathobiology of human neuroblastoma.     

Specific expression of the ret proto-oncogene in human neuroblastoma cell lines

The expression of the ret proto-oncogene (proto-ret), which possibly encodes two isoforms of a receptor-type tyrosine kinase, was examined in human tumor cell lines. Expression of the proto-ret mRNA was detected in all 11 neuroblastoma cell lines examined. The level of mRNA varied more than 100-fold in these neuroblastoma cell lines and was particularly high in three of them. On the other hand, 19 non-neuroblastoma tumor cell lines derived from solid tumors and a human diploid fibroblast cell line did not express any detectable levels of proto-ret mRNA. No remarkable amplification of the proto-ret or gross structural changes in the coding region were found in these neuroblastoma cell lines. The specific expression of the proto-ret in neuroblastomas suggests that the proto-ret product may have a role in cellular functions specific to neuroblastoma cells.     

Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo.     

Collagen biosynthesis in human neuroblastoma cell lines: evidence for expression of glial cell properties

An analysis was done on the synthesis of collagen, an extracellular matrix protein synthesized by Schwann cells, by several human neuroblastoma lines. Cultured cells were incubated in the presence of L-[2,3-3H]proline, and the collagens synthesized and secreted into the culture medium were analyzed by electrophoresis on acrylamide gels, ion exchange chromatography, and immunoprecipitation. The amount of collagen secreted by 4 cell lines tested represented less than 3% of the total protein synthesis, indicating a low degree of collagen biosynthesis by this tumor type. Analysis of collagen types secreted by all cell lines revealed the presence of a high-molecular-weight collagen precursor (Mr = 165,000) identified as type IV procollagen. In addition, several cell lines synthesized stromal type I and type III collagens. These studies show that neuroblastoma cells produce collagenous proteins including basement membrane collagen (type IV) and stromal collagens (types I and III), indicating that these cells express properties of glial cells such as Schwann cells.     

Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines

A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.     

Establishment and characterization of human neuroblastoma and ganglioneuroblastoma cell lines.

The establishment in culture and characterization of 4 human neuroblastoma (NB) cell lines and 1 human ganglioneuroblastoma cell line are described. Each cell line fulfilled at least 2 of 4 criteria for malignant or transformed cells: viz., subcultured more than 70 times, high saturation density with absence of contact inhibition, population-doubling time within a range of 10-40 h, and tumour formation in nu/nu mice. Each cell line also fulfilled at least 2 of 3 criteria for neuroblastoma cells: viz., humoral and cell-mediated immune reactivity toward NB-associated cell-surface antigen, intracellular storage and extra-cellular secretion of catecholamines, and characteristic neuroblast and ganglion-cell morphology.     

Differential expression of bcl2 protooncogene in neuroblastoma and other human tumor cell lines of neural origin.

The bcl2 protooncogene was originally discovered because of its involvement in t(14;18) chromosomal translocations frequently found in non-Hodgkin's lymphomas. The expression of this gene is reported to be highly tissue specific, with bcl2 mRNAs being readily detectable only in hematolymphoid tissues and brain. To explore the possible involvement of bcl2 in neural tumors, we surveyed a variety of tumor cell lines for the presence of the p26-BCL2 protein by immunoprecipitation and immunoblotting methods. Very high levels of BCL2 protein were found in three of nine neuroblastoma (NB) cell lines examined; these levels of p26-BCL2 were comparable to lymphoma cell lines that contain a t(14;18). Despite the impressive relative amounts of BCL2 protein, however, no structural alterations or changes in the methylation status of bcl2 genes were detected in these NB cell lines by conventional Southern blotting. Of the other NB cell lines surveyed, three contained intermediate levels of BCL2 and another three cell lines had little or no detectable BCL2 protein, raising the possibility that determination of relative levels of BCL2 protein may help to segregate neuroblastomas into groups with different biological and clinical characteristics. BCL2 protein levels were not influenced by induction of neuronal differentiation with nerve growth factor in two of the two cell lines examined [SH-SY5Y (high BCL2); GICAN (low BCL2)] and did not correlate with N-MYC gene amplification or expression of nerve growth factor receptors. NB cell lines that contained little or no detectable BCL2 protein, however, tended to contain significant proportions of flat epithelioid cells, whereas bcl2-expressing cell lines were composed primarily of neuronal-like cells, suggesting that expression of this protooncogene correlates with the differentiation characteristics of these tumor cell lines. In addition to NBs, lower levels of BCL2 protein were also found in a variety of other neural crest-derived tumors and tumor cell lines, including some neuroepitheliomas, Ewing's sarcomas, neurofibromas, and melanomas. With regard to tumors of central nervous system origin, bcl2 expression was absent from most medulloblastomas but was detected at moderate to low levels in a retinoblastoma and some glioblastoma multiforme cell lines. Taken together, these findings imply that bcl2 protooncogene expression is differentially regulated within the various lineages of cells that give rise to the nervous system.     

Deferoxamine and human neuroblastoma and primitive neuroectodermal tumor cell lines.

Deferoxamine at concentrations of 3.28 microM to 32.8 microM for five days causes in vitro growth inhibitory and cytolytic activities in human neuroblastoma and neuroectodermal cell lines. These effects are most likely due to intracellular iron depletion and vary with each cell line tested. A 3.28 microM threshold for cytolytic effects was observed in the most sensitive cell lines SK-N-DZ and SK-PN-LI, while proportionate responses ranging from lysis to relative growth inhibition was observed in the more refractory VA-N-BR, SK-N-LO and SK-N-AS cell lines. Cytolytic effects may represent an artifact of the in vitro setting where maximum exposure of cells to the drug can be achieved. Different sensitivities to deferoxamine in controlled in vitro conditions suggest variable anti-tumor effects can be expected in the clinical setting. Deferoxamine in patients may require a maximum tolerated dosage as a constant infusion for greater than 72 hours.     

Similarities in glycosylation of human neuroblastoma tumors and cell lines

The presence of fucosyl residues linked alpha 1----3(4) to N-acetylglucosamine was demonstrated on the oligosaccharides from glycoproteins of 11 human neuroblastoma tumors from ten different patients. This finding is in complete agreement with the previous report that human neuroblastoma cell lines contained an unusually large proportion of metabolically incorporated L-[3H]fucose in this specific linkage (U. V. Santer and M. C. Glick, Cancer Res., 43:4159-4166, 1983). Furthermore, the glycopeptides derived from the neuroblastoma tumors had a low percentage of fucose-containing biantennary oligosaccharides as determined by affinity to concanavalin A-Sepharose and in this characteristic were similar to glycopeptides from virus transformed and other tumor cells. To obtain these results, the tumor cells were labeled metabolically for 48 h with L-[3H]fucose. The cells were harvested and digested with Pronase, and the glycopeptides were isolated and treated with alpha-L-fucosidase from almonds, specific for the release of fucose linked alpha 1----3(4) to N-acetylglucosamine. A portion of the glycopeptides was characterized by serial affinity chromatography on immobilized concanavalin A and lentil lectin. The phenotypic similarity of the tumor cells to the cell lines, particularly CHP-134, included the paucity of biantennary oligosaccharides and the presence of fucosyl residues on the multiantennae of the glycopeptides.     

Alpha-smooth-muscle actin and desmin expressions in human neuroblastoma cell lines

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth-muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural-crest tumor) exhibit at least 2 distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies (MAbs) against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha-smooth-muscle actin antibody. In addition, one of these lines (KP-N-SI) bound anti-desmin MAbs as determined by indirect immunofluorescence. A total of 8 cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to have the same neuroblastoma origin as each parent cell line by chromosomal analysis. Alpha-smooth-muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by 2-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3' UT) to human alpha-smooth-muscle actin mRNA. These ascertain the presence of alpha-smooth-muscle actin and desmin in neuroblastoma cell lines. These data show that, in addition to giving rise to cells with neural, Schwann-cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth-muscle cell markers.     

Expression of integrin receptors on 45 clinical neuroblastoma specimens

Immunohistological expression of integrins has been analyzed on 45 neuroblastoma specimens representative of the different clinical and histological forms of the tumor. None of the specimens expressed the alpha 5 chain of the integrins. The beta 1 chain was expressed on all specimens, the alpha 1 chain on 44 specimens and the alpha 3 chain on 42; the 4 specimens which lacked alpha 1 or alpha 3 were stage-4 neuroblastomas. The alpha 2 chain was expressed on 18 specimens, and the alpha 6 chain on 17; 15 reacted with both. Their reactivity was related to the maturation of the tumor rather than the stage of the disease: they were expressed on low-grade, well-differentiated specimens; stage 3-4 neuroblastoma specimens analyzed at diagnosis were negative, but usually expressed both chains when analyzed after in vivo differentiation by chemotherapy. alpha v reacted with 18 specimens and beta 3 with 12, without strict relation with the stage of the disease and/or its degree of differentiation; 9 well-differentiated specimens expressed the beta 4 chain; only 4 well-differentiated specimens expressed the alpha 4 chain. The 4 specimens which lacked alpha 1-beta 1 or alpha 3-beta 1 expression had n-myc amplification, whereas those which expressed either alpha 4, beta 4, beta 3 or alpha v had no amplification. Furthermore, the expression of the 3 heterodimers alpha 4-beta 1, alpha v-beta 3 and alpha 6-beta 4 was essentially observed on primary tumors which developed in the mediastinum. The expression of alpha 2-beta 1 and alpha 6-beta 1 was observed on both n-myc-positive and -negative specimens. beta 1 and alpha 3 were diffusely expressed on all counterparts of these tumors, from undifferentiated neuroblasts to ganglion and Schwann cells. The alpha 1 chain reacted with undifferentiated and intermediate neuroblasts as well as with Schwann cells, but ganglion cells were negative. alpha 2 and alpha 6 chains were negative on undifferentiated neuroblasts, variably expressed on intermediate neuroblasts, and restricted to Schwann cells in ganglioneuroma. The expression of alpha 4 and beta 4 was restricted to Schwann cells. alpha v and beta 3 occasionally reacted with undifferentiated and intermediate neuroblasts; alpha v was strongly positive on Schwann cells but negative on ganglion cells, whereas beta 3 was positive on both neuronal and non-neuronal populations.     

The radiation dose-rate effect in two human neuroblastoma cell lines

The current use of targeted radiotherapy in the treatment of neuroblastoma has generated a requirement for further information on the radiobiology of these cells. Here we report on studies of the dose-rate effect in two human neuroblastoma cell lines (HX138 and HX142) and the recovery that they demonstrate in split-dose experiments. The sensitivity of the two cell lines to high dose-rate irradiation was confirmed. Surviving fractions at 2 Gy were 0.083 for HX138 and 0.11 for HX142. There was little evidence of a dose-rate effect above 2 cGy min-1 but significant sparing was seen at lower dose rates. Substantial recovery was seen in split-dose experiments on both cell lines, to an extent that was consistent with the linear quadratic equation. The data were used to derive values for the beta parameter of the linear-quadratic equation; the values for the neuroblastomas were higher than for any of the other human tumour cell lines that we have investigated to date. Thus, despite their high sensitivity to ionising radiation HX138 and HX142 do exhibit substantial levels of cellular recovery, suggesting that they may have a significant capacity for repair of radiation-induced lesions.     

Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing.     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.     

Expression and distribution of peripherin protein in human neuroblastoma cell lines

A series of human neuroblastoma (NB) cell lines was analyzed for expression of peripherin, a class-III intermediate filament protein expressed at high levels in ganglia of the peripheral nervous system. By Western blotting, peripherin protein was detected in all human NB cell lines examined. The highest level of peripherin was found in the NUB-7 cell line, previously characterized as homogeneously neuroblastic. By immunofluorescence labeling, peripherin was shown to be organized in a perinuclear filamentous pattern and, exemplified by IMR32 cells, was also shown to be localized to spontaneously formed neurites. Peripherin was expressed in neuroblastic but not substrate-adherent cells, and was found at low levels in I-type cells. There was a pronounced redistribution of peripherin to neurites formed in response to dibutyryl cyclic adenosine monophosphate (dbcAMP) and all-trans-retinoic acid (RA). In NUB-7 cells, which do not extend neurites in response to nerve growth factor, there was no change in the level of peripherin protein following treatment with this agent. Both dbcAMP and RA induced a redistribution of peripherin to neurite extensions, but only treatment with RA increased the level of the protein as demonstrated with NUB-6A4 and NUB-6C4 subclones. Peripherin was also variably expressed in peripheral neuroepithelioma (NE) cell lines tested, but was organized into a more basket-like filamentous pattern in these cells. The heterogeneous expression and distribution of peripherin in NB and NE cell lines indicate that this protein is associated with maturation of the neuronal phenotype and hence serves as a differentiation marker for tumors derived from the neural crest.     

Schwannian cell differentiation of human neuroblastoma cell lines in vitro induced by bromodeoxyuridine

In normal development the neural crest gives rise to sympathetic neuroblasts, sensory and autonomic ganglia, as well as Schwann cells. One tumor arising from this tissue is the neuroblastoma (NB), a malignancy of the adrenergic component of the sympathetic nervous system. Recent histological studies have shown that neuroblastomas can present with a schwannian cell component, rich in S100 protein. We have investigated the differentiation of NB cell lines, GOTO and RT-LN-1, into a schwannian cell phenotype using bromodeoxyuridine (BrdU). This agent induced morphological changes in these cell lines. Flat-epithelial cells were identified in the GOTO cell line and both flat-epithelial and neuronal phenotypes were found in the RT-LN-1 cell line. S100 protein (beta-Subunit) was induced in both cell lines after 18-25 days of BrdU treatment as determined by enzyme-linked immunoassay. In addition increase in the beta-subunit of S100 protein was identified in BrdU-treated flat-epithelial cells by indirect immunofluorescence using a monoclonal antibody specific for the beta-subunit of the protein. Cyclic nucleotide phosphodiesterase activity significantly increased in both BrdU-treated NB cell lines, as compared with nontreated cells. However no significant increase of glial fibrillary acidic protein in BrdU-treated cells was found either by enzyme-linked immunoassay or indirect immunofluorescence using a monoclonal antibody to glial fibrillary acidic protein. Thus, cells with Schwann cell characteristics can clearly be identified in the neuroblastoma cell lines after BrdU treatment. Fluorescence-activated cell sorting analysis revealed no quantitative changes in cell membrane antigens recognized by monoclonal antibodies UJ-13A (neuroectodermal associated antigen) and anti-Thy-1 (Thy-1) on BrdU treatment. In contrast, UJ-127-11 (neuroectodermal associated) decreased, and W6/32 and BB7.7 (HLA-ABC) and BBM.1 (beta 2-microglobulin) markedly increased in both BrdU-treated cell lines. No induction of L243 (HLA-DR), B7/21 (HLA-DP), and Genox 3.55 (HLA-DQ) was noted. The increased HLA-ABC (HLA class I) antigen may enable BrdU-treated NB cells to be recognized by cytotoxic T-cells. This may be related to the pathological evidence that NB patients whose tumors are rich in S100 protein have a better prognosis. Further studies on the potential of differentiation agents to induce a phenotypic change, that is associated with an improved prognosis for NB patients, are required.     

The radiosensitivities of human neuroblastoma in vitro--a comparison of three cell lines

The radiation response of three human neuroblastoma cell lines (GOTO, NB1M, and SINCG) has been studied in vitro, using single cells both in the exponentially proliferative and the plateau phases. The endpoint used was clonogenic cell survivals. Cell survival curves, obtained from all cell lines, were characterized by small D0 values (0.4-0.55 Gy). However, only the GOTO cells showed the lack of a shoulder in the survival curve, whereas the other cell lines, especially the SJNCG, showed a broad sized shoulder. Delayed assay experiments with SJNCG cells showed larger repairs of potentially lethal damage, both in the exponentially proliferative and plateau phases, in contrast to GOTO cells. Further, the cell capacity for a split dose recovery was greater in SJNCG cells than in the GOTO cells. It has been said that hyperfractionation radiotherapy might be a better treatment for a neuroblastoma than the conventional therapy, however, we would suggest that a modification of the radiotherapy schedule ought to depend on the type of cells, because there are cells which show broad shoulder curves and a strong capacity for repair.     

Signal transduction in adherent and non-adherent human cell lines after fibronectin stimulation

It is shown that adherent and non-adherent human ovarian carcinoma cells (OVP 10) secrete MMPs and their production was stimulated by fibronectin as documented by gelatinise zymography. These cells also presented an increase of ERK phosphorylating activity following fibronectin stimulation, regardless of their adhesion. Contrary to OVP 10 cells, the human urothelial cells (HCV-29) are more anchorage-dependent. They only secreted the MMPs under adherent conditions and they revealed a lower level of basal and fibronectin stimulated ERK phosphorylation activity. In addition, non-adhering HCV-29 cells showed post translational down-regulation of focal adhesion kinase.