INVESTIGADORES
ALONSO Daniel Fernando
artículos
Título:
Optimization of molecular detection of GD2 synthase mRNA in retinoblastoma
Autor/es:
VE LAURENT; LL OTERO; V VAZQUEZ; S CAMARERO; MR GABRI; M LABRADA; DAVILA MT; G CHANTADA; DF ALONSO
Revista:
MOLECULAR MEDICINE REPORTS
Editorial:
SPANDIDOS PUBL LTD
Referencias:
Año: 2010 vol. 3 p. 253 - 259
ISSN:
1791-2997
Resumen:
Extraocular dissemination is the main cause of death in patients with
retinoblastoma (RB) in developing countries, and there are few molecular
markers that are useful for the evaluation of minimal disseminated
disease. The GD2 ganglioside is known to be expressed by RB cells that
metastasize in bone marrow, and the activity of the enzyme responsible
for its synthesis, GD2 synthase, can be detected in neuroblastoma, which
shares many phenotypic features with RB. The purpose of the present
study was to optimize the detection of GD2 synthase expression by
reverse transcription-polymerase chain reaction (RT-PCR) followed by
nested-PCR in human RB cell lines and patient samples. The optimization
strategy was carried out using the RB cell lines Y79 and WERI-Rb1 and
specific primers designed for the human sequence of GD2 synthase mRNA.
We detected GD2 synthase expression with at least 200 and 40 pg of total
RNA extracted from cultured RB cells using a first round of RT-PCR
amplification or a second round of nested-PCR, respectively. We also
confirmed the expression of GD2 synthase by RT-PCR and
immunohistochemical detection of the ganglioside in human RB tumors
xenotransplanted in nude mice. Using tumor bank specimens from eight RB
patients, we were able to demonstrate the presence of GD2 synthase mRNA
in blood and cerebrospinal fluid samples in cases of extraocular
dissemination of the tumor. The sequence was not detected in samples
derived from children with low-risk disease or healthy adult volunteers.
Hence, GD2 synthase mRNA detection through an optimized nested RT-PCR
assay is a promising tool for the assessment of minimal disseminated
disease in enucleated patients.