Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression (original) (raw)
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Cancer Letters, 1988
In the present study the effect of all-transretinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (I) The tumor mince was incubated with 1 fl RA for 30 min at 3OOC; the RNA polymerase actiuity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 2!S°C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the
Cellular and Molecular Neurobiology, 1990
1. Neuroblastoma (NB) is an unusual neuroectodermal tumor showing a high degree of spontaneous regression. NB cells can be induced to differentiate in vitro by various agents. Cell differentiation results in morphological changes characteristic of the mature neuronal phenotype, including outgrowth of neuritelike structures with several interconnections. 2. Recent experiments indicate that morphological differentiation of NB cells is associated with changes in expression of N-myc, c-myc, and c-myb oncogenes and synthesis of neurofilament proteins. However, little is known about the transcription of neurofilament genes during differentiation. 3. We have analyzed the expression of both the N-myc oncogene and mid-size neurofilament (NF) genes in the LAN-1 human NB cell line, cultured in the presence of retinoic acid (RA). Continuous treatment with RA induced morphological differentiation within 5-6 days. The transcription of N-myc was down-modulated within 24 hr of the initial exposure to RA. The mid-size NF mRNA was increased at this time. The expression of N-myc was not modified in serum-deprived LAN-1 cells, indicating that N-myc transcription is unaffected by the arrest of the cells in the G1 phase.
Proceedings of the National Academy of Sciences, 1984
Previous studies had revealed that DNA with partial similarity to the myc oncogene (N-myc) is frequently amplified in human neuroblastoma cell lines and neuroblastoma tumors. We show here for one patient that N-myc amplification is confined to the neuroblastoma tumor and is not present in normal tissue. N-myc mRNA -4.0 kilobases in size is detectable in neuroblastoma cell lines and tumors and in a retinoblastoma cell line. By contrast, appreciable amounts of this RNA were not present in a number of cell lines derived from other human tumors and in fibroblasts from a normal individual and from a neuroblastoma patient. Low levels of Nmyc RNA were found in human and murine neuroblastoma cell lines lacking amplification of this gene, up to 80-fold greater levels in all cell lines carrying amplified N-myc. In situ hybridization to sections of neuroblastoma tumors revealed high expression of N-myc predominantly in undifferentiated neuroblasts. We hypothesize that amplification and consequent elevated expression of N-myc may be related to malignant progression.
Proceedings of the …, 1989
retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion highperformance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and >95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RARa or RARE were enriched (20to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high affinity binding of RA and its benzoic acid analogs ChS5, Ch3O, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of -0.46 nM and 1400 ± 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RARa and RARf indicated that HL-60 cells
1994
carcinomas in patients with head and neck or lung cancer, and skin cancers in patients with xeroderma pigmentosum (7â€"12). To evaluate the potential application ofretinoids for the prevention and treatment of head and neck premalignancies and SCC,3 we investigated the effects of retinoids on the growth and differentiation of Cultured HNSCC cells (13â€"18). We found that BA inhibited the proliferation of the majority of the HNSCC cell lines we examined and suppressed their transformed phenotype (13, 15, 18). These findings contrasted with previous reports that BA enhanced the growth of normal buccal mucosa cells (19), suggesting a selective inhibition oftumor cells. In addition, BA suppressed squamous differentiation of HNSCC cells (14, 15, 17, 20) and modulated two differentiation pathways in normal laryngeal keratino cytes and papilloma cells (21, 22). The mechanism(s) by which retinoids suppress carcinogenesis and regulate differentiation and the expression of the transformed pheno type in malignant cells has not been elucidated. It is thought that nuclear retinoid receptors, members of the steroid/thyroid hormone! vitamin D receptor family that act as ligand-activated transacting transcription factors, mediate the effects of retinoids on gene expres sion and thereby alter the growth and differentiation of normal and tumor cells. There are two types of retinoid nuclear receptors, RARs and RXRs. Each of the receptor types has at least three subtypes,-a,-@, and-‘y (3, 23â€"26). The RARs bind BA and 9-cis RA, whereas the RXRs bind 9-cis BA but not BA. The RXRs and RARs form het erodimers before binding to specific DNA sequences characterized by direct repeats of (A/G)GCITCA separated by two or five nucleotides, although complex elements have also been identified (24â€"26).Other factors affecting the retinoid-signaling pathway are the CRABP-I and CRABP-II, which have been implicated in controlling the level of BA available for interaction with nuclear receptors by sequestering the retinoid or increasing its catabolism (27, 28). The study presented here was designed to examine the expression of BA-binding proteins and receptors in HNSCC cell lines and their relationship to cell differentiation and responses to BA. MATERIALS AND METHODS Cell CUltUreand BA Treatment Procedures. The fourHNSCCcell lines (Table 1) were grown in monolayer cultures in a 1:1 (v/v) mixture of Dulbec co's modified Eagle's medium and Ham's F12 medium containing 5% FBS at 37°C. BA was dissolved in DMSO at a concentration of 102 Mand was stored in the dark at â€"20°C in N2. Stock solutions were diluted to the appropriate concentration with growth medium. Control cultures received the same amount of DMSO as treated cultures. For growth inhibition studies, cells were grown for 7 days in the absence (control) or presence of 1 @M RA. Medium was 3 The abbreviations used are: SCC, squamous cell carcinoma; HNSCC, head and neck
Experimental Cell Research, 1997
if allowed to grow in suspension to form aggregates or We have examined the mechanism of regulation of parietal endoderm if treated with cyclic AMP-generat-rRNA synthesis in mouse F9 teratocarcinoma cells that ing agents . EC cells mimic early mammalian develwere induced to differentiate by retinoic acid and diopment and differentiation and are therefore a useful butyryl cAMP. Ribosomal RNA (rRNA) synthesis was model system for studying the regulation of gene exsignificantly reduced during differentiation of F9 cells pression that accompanies early embryogenesis [9][10][11]. into parietal endoderm cells. Nuclear run-on assay re-This is frequently mediated by transcription factors vealed that the rRNA gene transcription rates were that regulate differentiation-specific gene expression reduced in differentiated cells, and this phenomenon [12][13][16] as well as activate cloned viral gene promoters could be mimicked by in vitro transcription assay us- . These transcription factors are themselves ing nuclear extracts prepared from F9 stem and F9 subject to regulation that involves changes in their parietal endoderm cells. Analysis of the DNA-binding amounts or activities. The abnormal level of expression activities of two RNA polymerase I (pol I) transcription or altered function of these factors can lead to drastic factors E 1 BF/Ku and UBF revealed decreased affinity cellular abnormalities . Because retinoids are for their cognate recognition sequences. Immunoblot known to suppress growth and promote differentiation analysis showed a marked reduction in the amounts of embryonal carcinoma and other malignant cells such