George R. Molloy

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene.

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.

Rat Brain Creatine Kinase Messenger RNA Levels Are High in Primary Cultures of Brain Astrocytes and Oligodendrocytes and Low in Neurons

Abstract: Rat brain creatine kinase (CKB) gene expression is highest in the brain but is also detectable at lower levels in some other tissues. In the brain, the CKB enzyme is thought to be involved in the regeneration of ATP necessary for transport of ions and neurotransmitters. To understand the molecular events that lead to high CKB expression in the brain, we have determined the steady‐state levels of CKB mRNA in homogeneous cultures of primary rat brain astrocytes, oligodendrocytes, and neurons. Northern blot analysis showed that whereas the 1.4‐kb CKB mRNA was detectable in neurons, the level was about 17‐fold higher in oligodendrocytes and 15‐fold higher in astrocytes. The blots were hybridized with a CKB‐specific 32P‐antisense RNA probe, complementary to the 3’untranslated sequence of CKB, which hybridizes to CKB mRNA but not CKM mRNA. Also, the 5’and 3’ends of CKB mRNA from the glial cells were mapped, using exon‐specific antisense probes in the RNase‐protection assay, and were found to be the same in astrocytes and oligodendrocytes. This indicated that (a) the site of in vivo transcription initiation in astrocytes and oligodendrocytes was directed exclusively by the downstream, nonconcensus TTAA sequence at ‐25 bp in the CKB promoter that is also utilized by all other cell types that express CKB and (b) the 3’end of mature CKB mRNA was the same in astrocytes and oligodendrocytes. In addition, there was no detectable alternate splicing in exon 1, 2, or 8 of CKB mRNA in rat astrocytes and oligodendrocytes. Also, our studies showed that 1.4‐kb CKB mRNA is expressed in established C6 glioma cells at an intermediate level about threefold higher than that in primary neurons.

Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum: evidence for transcriptional regulation.

Transcription and accumulation of brain-type creatine kinase (CKB) mRNA and its protein was examined during postnatal development of rat brain cerebellum, the brain region containing highest CKB mRNA in the adult. CKB protein was extremely low at day 1, increased about 10-fold until week 4 and remained constant until week 10. This time course was paralleled by cerebellar CKB mRNA, which was also extremely low at day 1 and increased 5-fold during the first 3 weeks and then remained constant. High levels of CKB protein were also detected in cultured primary cerebellar granular neurons. Nuclear run-on assays directly showed that CKB mRNA accumulation during postnatal cerebellar development was due to increased transcription. When compared with cerebrum and whole brain, cerebellar CKB mRNA accumulation during postnatal development was temporally delayed. Analysis of myocyte enhancer factor (MEF)-2 and Sp1, factors known to initiate or sustain CKB transcription in tissues other than brain, revealed that MEF-2 in cerebellum was low at week 1 but increased 3.5-fold by week 7, while Sp1 remained unchanged. The increase in CKB protein during cerebellar postnatal development was coincident with that of the ubiquitous mitochondrial CK protein and mRNA, indicating that a functional phosphocreatine energy shuttle probably exists for efficient ATP regeneration in the cerebellum. This should be beneficial for the many energy-demanding requirements during cerebellar development, as indicated by the observed temporal co-expression of CKB with myelin basic protein, which is involved in axon myelination by oligodendrocytes.

Creatine kinase-B mRNA levels in brain regions from male and female rats

The creatine kinase-B (CKB) enzyme is proposed to have a pivotal role in the regeneration of ATP in the nervous system. In the present study, the steady-state levels of CKB mRNA were determined by RNase protection assay in seventeen separate brain regions obtained from rats during the initial interval of the light period or period of inactivity in rats. The antisense probe used specifically hybridizes to CKB mRNA and discriminates CKB from CKM mRNA. The results show that brain regions from Wistar rats differ in CKB mRNA content. Highest levels of CKB mRNA were detected in the male and female cerebellum. High levels of CKB mRNA were observed in the spinal cord, brain stem and its structures (medulla, pons and midbrain) and olfactory bulb of the male rats. Female rats also contained high levels of CKB mRNA in the brain stem. In both male and female rats, the frontal cortex, occipital cortex, hippocampus and striatum exhibited lower levels of CKB mRNA relative to the complete brain. Statistical analyses demonstrated a significant difference between the male and female CKB mRNA profiles. However, CKB mRNA levels in brain regions with estrogen receptors (hypothalamus, hippocampus) were similar in male and female rats. Differential CKB mRNA levels in various brain regions may suggest diverse physiological significance of the CKB system in the regulation of brain energy metabolism.

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

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.

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.

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 Gαs 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. J. Neurosci. Res. 56:371–385, 1999.

Effect of 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole on ribonucleotide metabolism and accumulation of mitochondrial RNA and low-molecular-weight cytoplasmic RNA in HeLa cells

These results provide additional information on the selective inhibition of RNA synthesis by 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (DRB). DRB only slightly inhibited the poly(A+) RNA and ribosomal RNA in the mitochondria (maximal inhibition was ~25%) but severely inhibited the poly(A+) RNA in the postmitochondrial supernatant (~95%) and the poly(A+) RNA associated with the cytoplasmic membranes (~80%). Separation of the cytoplasmic low-molecular-weight RNAs showed that DRB inhibited the 5.8 S rRNA, a product of RNA polymerase I, by ~95% while there was only a slight inhibition of the 4 S RNAs (~20%) and 5 S RNA (<5%), products of RNA polymerase III. DRB severely inhibited the appearance in the cytoplasm of 28 S rRNA (~95%) and 18 S rRNA (~80%). These results, along with other recent reports (31–34), may suggest that DRB most severely inhibits RNAs that are extensively processed and/or transcribed from genes that contain extensive intervening sequences. These experiments also indicate that the mechanism of DRB inhibition does not involve alterations in ribonucleotide metabolism. DRB did not affect the phosphorylation of any ribonucleotides to triphosphates or the cellular conversion of [3H]uridine to UTP. Also, the size of the UTP and ATP pools in DRB-treated cells was equal to or greater than those in control cells through a period of 240 min. Significant amounts of DRB triphosphate could not be detected in DRB-treated cells suggesting that this may not be the inhibitory form of DRB. Measurements of the specific activity of the UTP pool allowed direct measurements of the accumulation of picomoles of the individual RNAs in the presence of DRB.

Inhibition of messenger RNA synthesis by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole without its detectable accumulation in mouse T-lymphoma cells

Abstract Two lines of evidence were obtained which indicate that inhibition of mRNA formation does not require the detectable accumulation of 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB), a halogenated analog of adenosine. First, the extent of inhibition by DRB of the formation of cytoplasmic poly(A)+ RNA was as rapid and severe (>90% inhibition) in wild-type mouse lymphoma cells (S49) as in mutant cells (AE1), derived from S49, which were deficient in the transport of purine and pyrimidine nucleosides. Second, the accumulation of [3H]DRB was measured directly and compared to the accumulation of [3H]adenosine. Whereas S49 cells accumulated [3H]adenosine in a linear manner, neither S49 nor AE1 cells accumulated [3H]DRB to a significant extent. This suggests that inhibition of mRNA synthesis by DRB may (1) require the transport and intracellular accumulation of only minute amounts of DRB, or (2) result from secondary event(s) triggered by interaction of DRB with a surface membrane component.

Isolation of oligo(U)-containing heterogeneous nuclear RNA from control and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-treated HeLa cells

Abstract Most (54–79%) of the heterogeneous nuclear RNA (hnRNA) which contains oligo(U) sequences was specifically retained on columns of poly(A) Sepharose and separated from hnRNA which lacked oligo(U) sequences. The isolation of oligo(U)-containing hnRNA was maximized by treating the hnRNA with HCHO prior to chromatography. This permitted an initial characterization of the oligo(U)-containing hnRNA. Experiments suggest that HCHO denatured the hnRNA and rendered the oligo(U) sequences accessible to poly(A) Sepharose. In denatured hnRNA, the percentage of molecules which contained an oligo(U) sequence increased with the size of the hnRNA; 32–57% of the large hnRNA [8–13 kilobases (kb) long] contained an oligo(U) sequence while only 11–14% of the 2-kb-long hnRNA contained an oligo(U) sequence. The number of oligo(U) sequences per molecule was also measured in denatured hnRNA of varying length. While the largest hnRNA class analyzed (13 kb) was found to contain the highest percentage of oligo(U)-containing molecules (57%), the 8- and 2-kb-long hnRNA fractions contained a greater total number of oligo(U)-containing molecules. The percentage of hnRNA molecules which contained an oligo(U) sequence, the number of oligo(U) sequences per molecule, and the size of the oligo(U) sequence were similar in both control hnRNA and the fraction of hnRNA (~30%) which is resistant to inhibition by 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole.