Gene expression of neuronal nitric oxide synthase and adrenomedullin in human neuroblastoma using real-time PCR

Abstract

The objective of our study was to assess the gene expression of the antiproliferative systems neuronal nitric oxide synthase (nNOS) and adrenomedullin (AM) in human neuroblastoma. A novel real‐time PCR method was evaluated using neuropeptide Y (NPY) for validation. Glyceraldehyd‐3‐phospate dehydrogenase (GAPDH) and NPY gene expression in neuroblastomas of 50 patients were measured in parallel by competitive quantitative and TaqMan real‐time RT‐PCR. AM and nNOS mRNA were determined by real‐time PCR. Our results showed a linear relationship between competitive quantitative and real‐time RT‐PCR measurements of NPY and GAPDH (r = 0.87 and r = 0.92, respectively). AM and nNOS mRNA was found in all tumor samples. AM/GAPDH mRNA increased with higher differentiation according to Shimada (p = 0.013). There was no relation between MYCN amplification nor with the tumor stage (p = 0.78 and p = 0.30, respectively). AM/GAPDH did not relate to recurrence or death in a 5‐year follow‐up period. Neuronal NOS/GAPDH expression did not relate to any biological or clinical parameter of prognosis or differentiation. Similar results were obtained when the neuronal marker protein gene product 9.5 (PGP9.5) was used to normalize mRNA concentration. In conclusion, TaqMan real‐time PCR appears to be a reliable method to quantify gene expression in neuroblastomas. Adrenomedullin mRNA in neuroblastoma is linked to tumor differentiation but not to prognostic markers. Int. J. Cancer 88:172–175, 2000. © 2000 Wiley‐Liss, Inc.

Antiproliferative effect of nitric oxide on epidermal growth factor‐responsive human neuroblastoma cells

“…Previous work with other neuroblastoma cell lines have shown that some of these cells express nNOS (Fujisawa et al . 1994; Dotsch et al . 2000) and that, under treatment with differentiation inductors such as tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) (Muñoz‐Fernandez et al .…”

“…The small response to L-NAME, observed in serum-treated cells, correlates well with the low enzyme expression detected and also with the fact that, although both isoforms were present, only iNOS was probably active, as nNOS needs specific signals leading to intracytoplasmic increases in calcium concentration to synthesize NO from L-arginine (Moncada et al 1991). Previous work with other neuroblastoma cell lines have shown that some of these cells express nNOS (Fujisawa et al 1994;Dotsch et al 2000) and that, under treatment with differentiation inductors such as tumour necrosis factor-a (TNF-a) and interferon-c (IFN-c) (Muñoz-Fernandez et al 1994;Ogura and Esumi 1996;Obregon et al 1997), retinoic acid (Ghigo et al 1998) or nerve growth factor (NGF) (Peunova and Enikolopov 1995;Phung et al 1999), iNOS may also be expressed and cellular NO production can be increased. Under these conditions, NOS inhibitors lead to reversal of the differentiation process, whereas exogenous NO induces differentiation.…”

Function of neuronal nitric oxide synthase enzyme in temozolomide-induced damage of astrocytic tumor cells

“…RT-qPCR was carried out in a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The forward and reverse primers for nNOS were 5'-GGT GGA GAT CAA TAT CGC GGT T-3' and 5'-CCG GCA GCG GTA CTC ATT CT-3' , respectively (24). For the housekeeping gene, the GC-rich promoter binding protein 1 primers 5'-TCA CTT GAG GCA GAA CAC AGA-3' and 5'-AGC ACA TGT TTC ATC ATT TTC AC-3' were used (25).…”

Reliable Transcript Quantification by Real-Time Reverse Transcriptase-Polymerase Chain Reaction in Primary Neuroblastoma Using Normalization to Averaged Expression Levels of the Control Genes HPRT1 and SDHA

“…Quantification of target gene expression levels by realtime reverse transcriptase-polymerase chain reaction (RT-PCR) 1,2 is becoming increasingly significant in neuroblastoma as in other human malignancies to gain insight into the molecular pathology of the tumor, to identify novel prognostic markers, or to evaluate minimal residual disease response. [3][4][5][6][7][8][9] A common feature of most transcript quantification techniques is the requirement for normalization, because a number of variables may influence the results. 2,10 Ideally, abundances of mRNA levels are determined as transcript copies per cell.…”