Neuroblastoma (NB) is a very common malignant solid tumor in childhood. Prognosis in NB patients tends to vary greatly, and many studies have demonstrated that both clinical and molecular biological factors are correlated with outcome. 1) For example, patients under the age of 1 year at diagnosis usually have good prognoses, but those diagnosed over the age of 1 year have poor prognoses.2) Increased expression of the molecular biological factors MYCN, H-ras and trkA is well known in NB. [3][4][5][6][7][8][9][10][11] Recently, there has been great interest in apoptosis, or programmed cell death, the mechanism by which cells essentially suicide.12) Many inhibitors of apoptosis are known to contribute to tumorigenicity and increased spread of tumor cells.13) Survivin is a recently described member of the inhibitor of apoptosis protein (IAP) family.14) This gene exists on chromosome 17q and inhibits apoptosis by blocking the effects of caspase-9, which is activated in extrinsic and intrinsic pathways.14-17) Survivin is expressed in many malignant tumors, including breast, lung, stomach, colon and pancreatic cancers, bladder tumors, malignant lymphoma, and NB.18) It is not usually present in normal tissues and is rarely found in mature tissues.17) Thus, survivin expression is likely to be an important prognostic factor in tumor malignancy, and we considered that survivin mRNA expression would be useful in determining tumor malignancy and prognosis in NB.We therefore used reverse transcription-polymerase chain reaction (RT-PCR) to investigate the expression of survivin mRNA in NB cell lines, normal blood cell samples, and clinical NB tumor samples.Here, we describe how the degree of expression of survivin mRNA is a very useful prognostic indicator.
MATERIALS AND METHODSCell Lines, Clinical NB Tumor Samples, and Normal Blood Cell Samples Three NB cell lines 19,20) SK-N-SH, 20,21) and NB- 39 20) ) were examined. They were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 g/l sodium bicarbonate under 5% CO 2 at 37°C. Two normal adult blood cell samples and 13 clinical NB tumor samples were also examined. Two of the tumor samples were from recurrent tumors. These tumor tissues had been stored at Ϫ80°C since collection. The clinical diagnoses for these patients had been made by histopathology. Informed consent was obtained from all patients before the study began.RNA Extraction Total RNA from the three cell lines and 13 NB tumor samples was extracted with TRI ZOL reagent (Gibco BAL) by the acid-guanidium-phenol chloroform extraction method.22) Total RNA from the two normal blood cell samples was extracted with TRI ZOL LS Reagent (Gibco BAL) by acid-guanidium-phenol chloroform extraction method.
16)Reverse Transcription-Polymerase Chain Reaction For determination of survivin mRNA expression, total RNA (1 mg) was reverse-transcribed in a 10 ml reaction mixture with a first strand cDNA synthesis kit (Rever Tra-a-TM , Toyobo). RT was performed with Oligo...