Expression of the rat brain creatine kinase gene in C6 glioma cells

Wilson, Charlie D.; Parameswaran, B.; Molloy, George R.

doi:10.1002/jnr.490350111

Cited by 14 publications

(33 citation statements)

References 44 publications

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“…The level of RNase-resistant [32P] RNA probe was analyzed on 6% acrylamide-8M urea gels, autoradiographed and quantified by densitometric scanning of X-ray films and by liquid scintillation counting of excised RNA bands. To calculate the basal levels of CKB mRNA in different cell lines, the yield of RNase-resistant [32P]CKB probe was calculated per microgram of total cell RNA in each sample [Wilson et al, 1993] and also normal ized to the amount of 18S rRNA in the sample as determined by the RPA [Ilyin et al, 1996;Wilson et al, 1997); each normalization yielded similar conclusions. Fig.…”

Section: Rnase Protection Assaymentioning

confidence: 99%

“…CKB exon 2 and y-actin have been described [Molloy et al, 1992;Wilson et al, 1993]. The lengths of the full-length and RNase-rcsistant probes for CKB exon 2 and 3' UTS are described in figure 3 of Wilson et al [1997], To employ the greatest sensitivity in comparing the CKB mRNA levels in neuroblastomas with CKB in glial cells (see fig.…”

Section: M¡ I||mentioning

confidence: 99%

“…Isolation of total cellular RNA (using the guanidinium thiocyanate/CsCl procedure), quantification of RNA, RNAse protection assay (RPA) of CKB mRNA using the 3' UTS probe or the CKB exon 2 probe and RPA of y-actin mRNA were performed as described [Wilson et al, 1993: Molloy et al" 1992, For each RPA, an equal amount of total cell RNA from control or treated cells was used as indicated in the figure iegends. The level of RNase-resistant [32P] RNA probe was analyzed on 6% acrylamide-8M urea gels, autoradiographed and quantified by densitometric scanning of X-ray films and by liquid scintillation counting of excised RNA bands.…”

Section: Rnase Protection Assaymentioning

confidence: 99%

“…Cells were not allowed to exceed 70% confluence. Polylysine-coated plates were pre pared as described by Wilson et al [1993]. For serum-free culture conditions [Zimmer and Van Eldik, 1989] cells were adapted to PC-1 medium (Ventrex) for 2-4 days (according to the manufacturers' conditions) prior to treatment with forskolin.…”

Section: Aterials and M Ethodsmentioning

confidence: 99%

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Expression of the Brain Creatine Kinase Gene Is Low in Neuroblastoma Cell Lines

Wilson¹,

Shen²,

Molloy³

1997

Dev Neurosci

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The cytoplasmic creatine kinase (CKB) enzyme has a central role in the regeneration of ATP in the brain. We have shown previously that CKB mRNA levels in cultured primary rat brain astrocytes and oligodendrocytes are much higher than in primary neurons. It has been suggested that high CKB expression is essential for the energy-demanding functions of glial cells. Conversely, CKB may be repressed in most neuronal cells; however, CKB protein has previously been detected by immunohistochemistry in several distinct groups of neurons in the adult rodent brain. Presently, little is known of the factors responsible for the high CKB expression in glia and possible repression in neurons. In this report, we investigated if low CKB mRNA was characteristic of some established neuronal cell lines. CKB mRNA was found to be extremely low in mouse CI300 neuroblastomas NS20Y and N1E-115 but 10-fold higher in NG108-15, a hybrid cell composed of a Cl300 neuroblastoma and a rat C6 glioma. Since we showed NG108-15 contained only rat CKB mRNA transcribed from the C6 glioma CKB gene, expression of CKB mRNA may be a manifestation of a glial property in NG108-15 cells. However, CKB mRNA expression in NG108-15 appeared not to be fully activated since it was still 5-fold lower than in (parental) C6 glioma and 10-fold lower than in cellular RNA from either total rat brain or cultured primary astrocytes. When neuronal differentiation was increased in NS20Y and N1E-115 by treating cells with prostaglandin E₁ and theophylline, the extremely low CKB mRNA level was not significantly changed. In a comparative study, the CKB mRNA levels in NS20Y, N1E-115 and neuronal RT4-B8 and RT4-E5 cells (from the rat RT4 peripheral neurotumor) were at least 50-fold lower than that in C6 glioma and 100-fold lower than in cultured primary astrocytes. These cell lines may provide a system for the identification of factors involved in the possible repression of CKB in many neuronal cells.

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Section: Rnase Protection Assaymentioning

confidence: 99%

Section: M¡ I||mentioning

confidence: 99%

Section: Rnase Protection Assaymentioning

confidence: 99%

Section: Aterials and M Ethodsmentioning

confidence: 99%

See 2 more Smart Citations

Expression of the Brain Creatine Kinase Gene Is Low in Neuroblastoma Cell Lines

Wilson¹,

Shen²,

Molloy³

1997

Dev Neurosci

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“…Fractions were precipitated with 2 volumes of 100% ethanol, pellets were resuspended in EPS [10 mM EDTA, Pipes (pH 6.4), 0.5% SDS and 0.15 M NaCl], deproteinized with phenol/chloroform and RNA was precipitated in ethanol. RNA pellets were dissolved in sterile, RNase-free H 2 O and analyzed on a 1% agarose/HCHO gel to check RNA integrity [17,28]. One half of each fraction was assayed for CKB mRNA using RPA.…”

Section: Isolation Of Polyribosomesmentioning

confidence: 99%

Expression of Creatine Kinase Isoenzyme Genes during Postnatal Development of Rat Brain Cerebrum: Evidence for Posttranscriptional Regulation

Shen¹,

Willis²,

Zhang³

et al. 2003

Dev Neurosci

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Brain creatine kinase (CKB) has a central role in the regeneration of ATP in the brain. During postnatal development of rat brain cerebrum, the CKB protein level was very low at postnatal day 1 and week 1 but by week 4 had increased 6- to 7-fold and remained constant through week 10. Surprisingly, CKB mRNA levels were already maximal at postnatal day 1 and week 1, indicating that CKB protein expression does not simply reflect the levels of CKB mRNA and is likely regulated posttranscriptionally during early postnatal times. Interestingly, the majority of cytoplasmic CKB mRNA was found to be associated with polyribosomes both at postnatal day 3 and week 6. Therefore, low CKB protein levels at early postnatal times could either be due to (1) normal translation initiation of CKB mRNA followed by a subsequent arrest during elongation or termination and/or (2) normal translation of CKB mRNA followed by rapid degradation of CKB protein. However, CKB protein increased coincidently with ubiquitous mitochondrial CK protein, suggesting that a functional phosphocreatine energy shuttle is formed in the cerebrum during postnatal development. The apparent posttranscriptional regulation of CKB in early postnatal cerebrum contrasts with the transcriptional regulation controlling accumulation of CKB protein in postnatal developing cerebellum.

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Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP-mediated increased transcription in glioblastoma cells

Kuzhikandathil

Molloy

1999

J. Neurosci. Res.

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Our previous studies have shown that transcription of brain creatine kinase (CKB) mRNA in U87-MG glioblastoma cells is stimulated by a forskolin-mediated increase in cyclic AMP (cAMP) via a pathway involving protein kinase A (PKA) and the activation of Galphas proteins. In this report, we have employed transient transfection to investigate the rat CKB gene elements essential for the cAMP-mediated induction of rat CKB transcription in human U87 cells and have mapped the transcription start site of the induced CKB transcripts. We found that the level of induced transcription from the transfected genomic rat CKB gene was the same whether transcription was driven by 2.9 kb of CKB promoter plus 5' flanking sequence or the 0.2 kb CKB promoter, suggesting that the proximal CKB promoter was essential. Also, the level of induced transcription of the chloramphenicol acetyl transferase (CAT) reporter gene driven by the 2.9 kb CKB promoter was the same as with the 0.2 kb CKB promoter. Analyses of a series of 5' deletions of the 0.2 kb proximal CKB promoter showed that the sequences between -80 bp and +1 bp were essential for the cAMP-mediated induction of CKB transcription, despite the absence of a consensus cAMP response element (CRE) sequence in that region. In agreement, gel mobility shift assays showed that nuclear extracts from U87 cells contained a protein(s) which bound specifically to a [32P]CKB DNA probe containing the -60 bp to +1 bp sequence. Mapping the 5' end of the CKB transcripts showed that the initiation of the cAMP-induced transcription occurred almost exclusively from the downstream transcription start site, apparently under the initiation direction of the nonconsensus (-28) TTAA element and not the consensus (-60) TATAAATA element. The results are discussed with regard to nuclear protein factors which may be involved, and the possible cAMP-mediated increase in CKB transcription during myelinogenesis, since the differentiation of oligodendrocytes has previously been shown to be accelerated by increased intracellular cAMP.

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Expression of the rat brain creatine kinase gene in C6 glioma cells

Cited by 14 publications

References 44 publications

Expression of the Brain Creatine Kinase Gene Is Low in Neuroblastoma Cell Lines

Expression of the Brain Creatine Kinase Gene Is Low in Neuroblastoma Cell Lines

Expression of Creatine Kinase Isoenzyme Genes during Postnatal Development of Rat Brain Cerebrum: Evidence for Posttranscriptional Regulation

Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP-mediated increased transcription in glioblastoma cells

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