Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.

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Expression of the rat glial fibrillary acidic protein (GFAP) gene is responsive to the intracellular level of cAMP. We have examined the sequence 5'-upstream of the transcription start site of the rat GFAP-encoding gene to determine the elements responsible for regulating the cAMP response. The RT4 cell lines consist of a neural stem-cell type RT4-AC and its three derivative cell types, one glial-cell type, RT4-D, and two neuronal-cell types, RT4-B and RT4-E. GFAP is expressed in the stem-cell type and the glial-cell type but is not expressed in the neuronal-cell types. Luciferase expression vectors containing various areas of the 10.8-kb region upstream ofthe transcription start site ofthe GFAP gene were transiently transfected into these RT4 cells. The effect of cAMP was examined by quantitating the transient expression of luciferase. We found that (s) the 5'-upstream region alone (up to 10.8 kb) allows expression of the GFAP gene in the stem-cell type, the glial-cell type, and a neuronal-cell type; (it) there are negative and positive cAMP-responsive elements that are juxtaposed within the region between -240 bp and -110 bp upstream and are functional in the stem-cell and glial-cell types but are not functional in the neuronal-cell type RT4-E; (Mi) there may be elements that respond to dibutyryl-cAMP in all three RT4 cell types within the region from 2 kb to 10.8 kb upstream of the transcription start site; and (iv) a regulatory luciferase plasmid pRLgfap-1, containing both the upstream and downstream regulatory regions of the GFAP gene, not only expresses luciferase but also responds to forskolin in the stem-cell type and the glial-cell type. This regulatory plasmid, however, does not express in the neuronal-cell type with or without the forskolin treatment.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Glial-specific cAMP response of the glial fibrillary acidic protein gene cell lines.

Kaneko

Hagiwara

Leader

et al. 1994

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“…8-10; N. Hagiwara, S. Imada, and N.S., unpublished work). The two neuronal types, RT4-B and RT4-E, exhibit only neuronal characteristics, including the expression of voltage-dependent Na+ and K+ channels and extension of neurite-like processes (7,11,12). The parental stem-cell type, RT4-AC, expresses both glial and neuronal phenotypes (7,12).…”

mentioning

confidence: 99%

Tissue-specific versus cell type-specific expression of the glial fibrillary acidic protein.

Kaneko

Sueoka

1993

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Expression of the glial cell-specific gene encoding glial fibrillary acidic protein (GFAP) is regulated in a tissue-specific (neural tissue versus other tissues) as well as a cell type-specific (glial cell versus neuron) manner. Using a family of rat neurotumor RT4 cell lines in which neuronal/glial differentiation occurs in vitro, along with cell lines of different tissue origins, we identified by transient- and permanent-transfection assays two negative regulatory regions, GFAP downstream regulators 1 and 2 (GDR1 and GDR2). Both regions lie 3' of the transcription start site; GDR1 is in a 2.7-kb region extending from the first intron through the fifth exon, and GDR2 is within 1.7 kb 3' of the polyadenylylation site. GDR1 alone is responsible for tissue-specific expression (suppression in nonneural tissues), while both GDR1 and GDR2 are necessary for cell type-specific expression (suppression in neuronal cells).

“…Our laboratory has described the isolation and characterization of clonal cell lines from the peripheral nervous system (PNS) tumor RT4 (1)(2)(3)(4)(5)(6)(7)(8). The RT4 tumor was induced by ethylnitrosourea injection of a newborn BDIX rat, a procedure that predominantly gives rise to PNS tumors (1).…”

Section: Introductionmentioning

confidence: 99%

Induction and segregation of glial intermediate filament expression in the RT4 family of peripheral nervous system cell lines.

Freeman¹,

Sueoka²

1987

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We have found glial fibrillary acidic protein (GFAP), the major component of astrocyte intermediate fiaments, to be expressed in cell lines of the RT4 peripheral neurotumor family. The RT4 family is a "stem-cell-like" cell line, RT4-AC, that spontaneously undergoes differentiation in culture to three derivative cell types. This process, termed cell-type conversion, results in a segregation among the derivative cell types of parental cell phenotypes that have been described as neuronal-like or glial-like. We have identified a 50-kDa GFAP-immunoreactive cytoskeletal protein and GFAP mRNA in continuous RT4-AC and RT4-D (glial-type derivative) cell lines, but not in two presumptive neuronal-type cell lines. This result suggests that GFAP gene expression is coordinately coupled with the expression of other glial properties during cell-type conversion. In addition, the RT4-AC and RT4-D sublines were found to significantly express GFAP only at high cell densities and not during logarithmic growth and to express GFAP precociously during morphological differentiation following treatment with 1 mM N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate. These observations closely reflect reports of glial filament expression in astrocyte cultures, suggesting that a common regulatory mechanism is employed by central and peripheral nervous system glia.Our laboratory has described the isolation and characterization of clonal cell lines from the peripheral nervous system (PNS) tumor RT4 (1-8). The RT4 tumor was induced by ethylnitrosourea injection of a newborn BDIX rat, a procedure that predominantly gives rise to PNS tumors (1). An unstable cell line, termed RT4-AC, has been described (1, 3) that expresses the glial marker protein S-100 and that also displays voltage-sensitive Na' and K+ channels. The RT4-AC cell line was described as a "stem-cell-type" line because of its ability to repeatedly give rise to three distinct progeny cell types in culture (Fig. 1). These derivative cell types have been described as glial-like (the RT4-D cell type), based on the expression of S-100 and on the loss of the excitable membrane phenotype, or neuronal-like (the RT4-B and RT4-E cell types), based on the loss of S-100 expression and on the retention and enhancement of the excitable membrane phenotype. This differentiation phenomenon has been termed cell-type conversion, and the apparent coordinate segregation of "glial" or "neuronal" properties with conversion has been termed conversion coupling. Clonal RT4 cell lines corresponding to these four cell types have been passaged continuously in culture for over 5 years.Droms and Sueoka (8) have shown that a normally unexpressed high-affinity y-aminobutyric acid uptake mechanism, generally considered to be a glial property in the PNS, can be induced in the RT4-D cell line by a combination treatment with N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) and testololactone, a synthetic androgen. Under the conditions described, the high-affinity y-aminobutyric acid uptake mecha...