Gapdh acts as an inducible not constitutive gene in cultured endothelial cells

Rimarachin, Julio A.; Norcross, J.J.; Szabo, Paul; Weksler, Babette B.

doi:10.1007/bf02631056

Cited by 8 publications

(5 citation statements)

References 15 publications

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“…The concentration of GAPDH mRNA is often used for normalization of nonuniform gel loading [22,46]. However, this nominally constitutively expressed glycolytic enzyme has been found to be stimulated in liver and adipose tissue by insulin [47], and in vascular endothelial cells by serum [46].…”

Section: Discussionmentioning

confidence: 99%

Dual Actions of Phorbol Ester on Cytochrome P450 Cholesterol Side-Chain Cleavage Messenger Ribonucleic Acid Accumulation in Porcine Granulosa Cells1

Lahav¹,

Garmey²,

Shupnik³

et al. 1995

Biology of Reproduction

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In earlier studies in cultures of porcine granulosa cells prepared from small antral follicles, steroidogenesis-related loci were inhibited by treatment for 48 h with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC). In the present investigation, cells were incubated in serum-free medium for 48 h, with various agents present during the last 2-24 h. With TPA at 30 ng/ml, the FSH-stimulated cAMP accumulation was markedly enhanced at all time points. FSH increased the concentration of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA throughout the 24-h incubation. At 4 and 8 h, TPA increased the accumulation of P450scc mRNA, having an additive effect with FSH. However, at 24 h, TPA markedly suppressed the FSH-induced increased in P450scc mRNA. Pretreatment of cells with FSH did not shorten the time required for TPA to become inhibitory. The stimulatory effect of 8-bromo-cAMP on P450scc mRNA also was augmented by TPA at 4 h, but significant inhibition was not observed at 24 h. The concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA, intended to be used for correction of gel loading, was stably increased by both cAMP and TPA. These effects of TPA suggest multiple actions of PKC(s) on the regulation of P450scc expression and other endpoints in ovarian granulosa cells.

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Section: Discussionmentioning

confidence: 99%

Dual Actions of Phorbol Ester on Cytochrome P450 Cholesterol Side-Chain Cleavage Messenger Ribonucleic Acid Accumulation in Porcine Granulosa Cells1

Lahav¹,

Garmey²,

Shupnik³

et al. 1995

Biology of Reproduction

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show abstract

“…Similarly, the calcium ionophore A23187 induces GAPDH transcription through specific motifs in its promoter (Chao et al 1990). Growth hormone (Freyschuss et al 1994), vitamin D (Desprez et al 1992), oxidative stress (Ito et al 1996), hypoxia (Graven et al 1994, Zhong & Simons 1999, manganese (Hazell et al 1999) and the tumour suppressor TP53 (Chen et al 1999), have all been shown to activate its transcription, which is also inducible in endothelial cells (Rimarachin et al 1992) and during apoptosis (Ishitani et al 1997). Furthermore, it has been shown to be upregulated in cancer -for example in rat hepatomas (Chang et al 1998), malignant murine cell lines (Bhatia et al 1994) and human prostate carcinoma (Ripple & Wilding 1995).…”

Section: Gapdhmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,430

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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“…However, during the study, increases in G3PDH mRNA were observed, from which it was concluded that it was not suitable. Previous reports indicate that G3PDH can behave as an "early response" gene in human endothelial cells in vitro after addition of fresh medium containing serum 19) . In addition, mRNA expression for G3PDH increases in the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated epidermal cells at a time correlating to hyperproliferation 20) .…”

Section: Fig 11 Detection Of C-jun Mrna Using Reverse Transcriptionmentioning

confidence: 99%

c-jun mRNA Expression and Profilin mRNA Amplification in Rat Alveolar Macrophages Exposed to Volcanic Ash and Sulfur Dioxide

et al. 2003

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Local residents exposed to heavy falls of ash discharged by Mt. Sakurajima, an active volcano, have been reported to develop acute and chronic inflammation of the respiratory tract. The present study aimed to determine the primary cause of this inflammation using an experimental model. Wistar rats were exposed for 5 days (4 h/d) to air containing 100 mg/m 3 volcanic ash (mass median aerodynamic diameter, 4.3 µm; geometric standard deviation, 1.7) with or without 1.5 ppm sulfur dioxide (SO 2 ). The lungs were then lavaged, and mRNA was extracted from alveolar macrophages and assessed by reverse transcription-polymerase chain reaction (RT-PCR). In the lavage fluid, no change in cellularity or increase in the content of tumor necrosis factor (TNF)-α α α α α was detected. However, at 1 h following exposure, 80% of macrophages were seen to have phagocytosed the volcanic ash. This percentage was unchanged at 24 h after exposure. Profilin mRNA content of the macrophages was elevated, and c-jun mRNA was expressed. Alveolar macrophages exposed to volcanic ash and SO 2 , therefore, are likely to have some inflammatory and fibrogenic potential.

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Gapdh acts as an inducible not constitutive gene in cultured endothelial cells

Cited by 8 publications

References 15 publications

Dual Actions of Phorbol Ester on Cytochrome P450 Cholesterol Side-Chain Cleavage Messenger Ribonucleic Acid Accumulation in Porcine Granulosa Cells1

Dual Actions of Phorbol Ester on Cytochrome P450 Cholesterol Side-Chain Cleavage Messenger Ribonucleic Acid Accumulation in Porcine Granulosa Cells1

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

c-jun mRNA Expression and Profilin mRNA Amplification in Rat Alveolar Macrophages Exposed to Volcanic Ash and Sulfur Dioxide

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