Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

Barabás, Éva; Nagy, K.; Várkonyi, V; Horváth, Attila

doi:10.1590/s0100-879x2001001000006

Cited by 8 publications

(9 citation statements)

References 11 publications

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“…The expression levels of GTP, GCS, OPase, GLNase, and GTF transcripts in gut mucosa and tumors were determined by a semiquantitative RT-PCR as described elsewhere (27,28). Briefly, total RNA isolated using RNeasy Mini Kit (Qiagen, Chatsworth, CA) was reverse transcribed using Ready-To-Go You-Prime First-Strand Beads (Amersham, Piscataway, NJ) and oligo (dT; Promega, Madison, WI).…”

Section: Rna Extraction and Semiquantitative Reverse Transcription Pomentioning

confidence: 99%

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

Kaufmann

Тодорова

Luo

et al. 2008

Nutrition and Cancer

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Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in gamma-glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the gamma-glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of gamma-glutamyl transpeptidase (GTP), gamma-glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), gamma-glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

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Section: Rna Extraction and Semiquantitative Reverse Transcription Pomentioning

confidence: 99%

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

Kaufmann

Тодорова

Luo

et al. 2008

Nutrition and Cancer

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“…Total RNA from tissue samples was extracted using Trizol reagent (Life Technologies Inc.) following the manufacturer's protocol and the semiquantitative RT-PCR was then carried out (Barabas et al, 2001). Briefly, the RNA isolated (10 mg) was reverse transcribed to cDNA in a 25 ml reaction mix containing 200 U MMLV reverse transcriptase (Life Technologies Inc.), 2 mg random hexamer (New England BioLabs), 6 U RNA guard (Amersham) and 100 mM dNTP mix at 371C for 1 h. The enzyme was inactivated at 901C for 4 min and quickchilled.…”

Section: Semiquantitative Rt-pcrmentioning

confidence: 99%

NF-κB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix

et al. 2003

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We demonstrate, for the first time, that the transcription factor NF-jB is constitutively activated during human cervical cancer progression. Immunohistochemical analysis was done using 106 paraffin-embedded cervical tissue specimens of different histological grades. In normal cervical tissue and low-grade squamous intraepithelial lesions, p50, RelA and IjB-a were mainly localized in the cytosol, whereas in high-grade lesions and squamous cell carcinomas, p50-RelA heterodimers translocated into the nucleus with a concurrent decrease in IjB-a protein. By Western blot analysis, p50 and RelA were detectable mainly in the cytosolic and nuclear extracts in normal and cancer tissues, respectively, and cytosolic IjB-a expression was detectable in normal but not in cancer cervical tissues. NF-jB DNA-binding activity increased during cervical cancer progression and the binding complex was mainly composed of the p50-RelA heterodimers as revealed by electrophoretic mobility shift assays. Semiquantitative RT-PCR analysis, however, showed increased levels of IjB-a mRNA in cancer samples presumably because of feedback regulation as a result of enhanced NF-jB DNA-binding activity and a consequent functional activation of NF-jB. Further immunohistochemical analysis with an antibody to phospho IjB-a revealed that phosphorylation occurs mainly in squamous intraepithelial lesions, suggesting that the IjB-a gets phosphorylated initially and degraded as the tumor progressed.

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“…The expression levels of the Bcl-2, Bax and caspase-3 transcripts were assessed by a relative (semiquantitative) RT-PCR following the recommendations in several recent publications [37][38][39]. Total RNA (1 lg), isolated by RNeasy Mini Kit (Qiagen, Chatsworth, CA) was reverse transcribed using Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech Inc, Piscataway, NJ) and oligo (dT) (Promega, Madison, WI).…”

Section: Rna Extraction and Detection Of Gene Products By Relative (Smentioning

confidence: 99%

Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats

Тодорова

Harms

Kaufmann

et al. 2004

Breast Cancer Res Treat

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Glutamine (GLN) is a non-essential amino acid that is present in nearly every biochemical pathway and is the major intraorgan nitrogen carrier. GLN via glutamate, is one of the precursors for the synthesis of glutathione (GSH), the major endogenous antioxidant in mammalian cells, which protects them from oxidative injury and cell death. Cancer cells have higher GSH levels than the surrounding normal cells, which attributes to a higher rate of cell proliferation and resistance to chemotherapy. Therefore, selective tumor depletion of GSH presents a promising strategy in cancer treatment. Experimental studies have associated decreased GSH levels with inhibition of proliferation and stimulation of apoptosis. Previous results of our laboratory have provided evidence that dietary GLN diminished tumor development in implantable as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer and elevated GSH in the host tissues. In this study we examined the effects of GLN on GSH levels in DMBA-induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in tumor cells. The results have shown that GLN supplementation caused a significant decrease in the tumor GSH levels and the ratio GSH/oxidized GSH (GSSG), accompanied by up-regulation of Bax and caspase-3, and down-regulation of Bcl-2. These findings suggest that dietary GLN supplementation suppresses mammary carcinogenesis by activation of apoptosis in tumor cells and this probably is a result of GSH down-regulation.

show abstract

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

Cited by 8 publications

References 11 publications

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

NF-κB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix

Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats

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