Neil Sidell

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alternations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.

Antiproliferative effect of the garlic compound S-allyl cysteine on human neuroblastoma cells in vitro

A variety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. In the current study, we have assessed the ability of S-allyl cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was observed in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 micrograms/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.

Neuroblastoma: clinical perspectives, monoclonal antibodies, and retinoic acid.

Neuroblastoma, the second commonest solid tumor in children, is a neoplasm of the peripheral autonomic nervous system that usually occurs before children are 6 years old. Therapy of localized tumor (clinical stage I and II) and of a special form of metastatic tumor (clinical stage IV-S) is usually successful, but treatment of widespread regional (clinical stage III) or metastatic (clinical stage IV) neuroblastoma is almost uniformly unsuccessful. Unfortunately, two thirds of children have stage III or IV disease at diagnosis. Several clinical trials are in progress. Preclinical investigations with monoclonal antibodies and retinoic acid may lead to new therapies. Monoclonal antibodies that react relatively selectively with neuroblastoma cells may be useful for diagnosis and therapy. Neuroblastoma cells have cytoplasmic retinoic-acid-binding proteins; treatment of cultured cells with retinoic acid induces morphologic maturation and markedly inhibits proliferation.

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.

Retinoic acid upregulates interleukin-2 receptors on activated human thymocytes

It has previously been demonstrated that retinoic acid (RA) enhances the blastogenic responses of human thymocytes. We have now delineated the cellular mechanism of this activity. When RA was added to resting thymocyte cultures in the presence of recombinant interleukin-2 (rIL-2), blastogenesis was increased two- to fourfold. By assessing the proportion of cells that became Tac-positive and showed DNA synthesis early in the activation process, we determined that the augmentation by RA was not caused by an increased recruitment of resting cells that are activated to undergo blast transformation. Instead, RA markedly potentiated the growth rate of long-term rIL-2-dependent thymocyte blasts and, correspondingly, increased the Tac expression on these proliferating cells. Thus, RA enhancement of thymocyte responses appears to be mediated by an increase in IL-2-receptor expression on thymocyte blasts, resulting in augmented IL-2-dependent growth. This effect is independent of the original activating stimulus since enhancement of thymocyte responses to phytohemagglutinin (PHA) was also shown to be caused solely by increased proliferation of IL-2-dependent blast growth. In contrast to these effects on thymocytes, peripheral blood lymphocyte (PBL) proliferative responses were unaffected by RA treatment and, correspondingly, RA affected neither IL-2 receptor expression on PBL blasts nor the growth of these cells. Taken together, the results of this study suggest that RA can modulate IL-2-dependent immune responses, in part, by upregulating the expression of IL-2 receptors on proliferating T lymphoblasts generated from cells at restricted stages of development.

Retinoic acid synthesis in normal and alzheimer diseased brain and human neural cells

Retinoids play fundamental roles in CNS development, but their distribution, metabolism, and function within the mature human CNS are unknown. In these studies, extracts of autopsy tissues recovered from histopathologically confirmed control and Alzheimer diseased brains were tested for their ability to synthesize retinoic acid. Retinaldehyde dehydrogenase (RLDH), the enzyme that forms retinoic acid from retinaldehyde, was present in hippocampus, frontal cortex, and parietal cortex. The RLDH activity of hippocampus and parietal cortex from Alzheimer diseased brains was 1.5- to 2-fold higher (p < 0.05) compared to the controls. In contrast, the RLDH activity of frontal cortex was the same for both Alzheimer diseased and control groups. A cultured human glioblastoma (U251) and neuroblastoma (LA-N-5) cell line synthesized retinoic acid from retinaldehyde or retinol, suggesting that a variety of neural cell types possess this activity. LA-N-5 cells grown in vitamin A-depleted medium had higher (p < 0.05) RLDH activity (0.35 +/- 0.04 nmol/mg/h) than LA-N-5 cells grown in vitamin A-replete media (0.15 +/- 0.02 nmol/mg/h). This difference was lost when retinol was added back to the medium, confirming that a reduction in vitamin A supply can induce RLDH activity in neural cells. However, this feedback mechanism does not appear to explain the higher RLDH activity of Alzheimer diseased hippocampus and parietal cortex, because the overall vitamin A status as indicated by serum retinol and carotenoid levels and by hippocampal retinoid content was similar for the Alzheimer diseased and control groups. These studies establish the presence of retinoids and RLDH activity in human brain tissues, and indicate that retinoic acid synthesis is modulated in some regions of Alzheimer diseased brain.

Retinoic Acid-Induced Differentiation of Human Neuroblastoma: A Cell Variant System Showing Two Distinct Responses

Retinoic acid (RA) has been shown to induce the differentiation of human neuroblastoma cells in vitro. In this study, we describe two variants of the SK-N-SH human neuroblastoma cell line that have dramatically different responses to RA. RA induces neuronal-like differentiation characterized by extensive neurite outgrowth, thick neurite bundles, and large cellular aggregates of SK-N-SH-N (SH-N) cells. In contrast, RA treatment of SK-N-SH-F (SH-F) cultures transforms the small neuroblast cells into large flattened, fibroblastic or epithelial-like cells. Karyotype analysis verified that the SH-N and SH-F cultures were derived from a common precursor cell. Confirmation of their markedly different responses to RA was obtained by metabolic labelling of glycoproteins and SDS-PAGE analysis. While both sublines showed very similar Coomassie-labelled protein bands and glycoprotein profiles in control cultures, dramatic differences between the lines were revealed following RA treatment. In contrast to their similar protein profiles, untreated SH-N and SH-F cells had quite different patterns of ganglioside biosynthesis in that GM3 was detected in SH-F cells but not in SH-N, while GM1 was only detected in SH-N. Cellular RA binding protein (CRABP) was detected in both SH-F and SH-N cells and their RA-transformed derivatives. These results demonstrate heterogeneity in the response to RA of neuroblastoma cells derived from a common origin that cannot be accounted for by differences in CRABP content. The SH-N and SH-F neuroblastoma sublines should provide a useful system for further studies of the molecular processes through which RA exerts its differentiation-inducing activity on this type of tumor.

Lymphocyte surface markers and cytotoxicity following cryopreservation

The effects of cryopreservation (CP) on lymphocyte subpopulation distribution and functional activity in blastogenic and cytotoxicity assays were tested. Peripheral blood lymphocytes (PBL) from 12 healthy human donors were obtained by Ficoll-Hypaque separation. Half of each sample was tested fresh, while the other half was cryopreserved and then thawed and tested the same day. Each sample of CP-PBL was compared to fresh PBL from the same donor in simultaneous assays. Following CP there was a significant reduction in the percentage of E, EA gamma, and EA mu rosette-forming cells with a reciprocal increase in EAC rosette-forming cells. The blastogenic response to alloantigens was stable following CP while blastogenesis in unstimulated control cultures was significantly reduced. Mixed lymphocyte culture (MLC)-induced cell-mediated lympholysis (CML) was consistently and significantly diminished by CP. Cytotoxicity in 4 h chromium release NK (K562), ADCC, and LDCC assays was also significantly diminished by CP. In contrast, cytotoxicity was unaffected in an 18 h cytotoxicity assay against adherent cultured melanoma target cells.

Augmentation of Human Thymocyte Proliferative Responses by Retinoic Acid

When 10(-5) to 10(-7) M all-trans retinoic acid (RA) was added to human thymocyte or tonsil lymphocyte cultures in the presence of mitogens or allogeneic stimulator cells, blastogenesis was increased up to 2.5-fold. No augmentation in proliferative responses of peripheral blood or spleen lymphocytes was observed. In the thymus, lymphocytes in the subclass of cells that did not bind peanut agglutinin (PNA) were responsible for the RA-induced enhancement. RA also increased the number of mitogen-stimulated thymocyte colonies developing in soft agar. These results indicate that the targets of RA activity must be lymphoid cells at a later stage of maturation than those identified by binding to PNA, and that one mechanism of RA enhancement is an increase in the number of lymphocytes that undergo blast transformation.