Trks Gene Expression In Human Neuroblastomas And The Effect Of K252a On Neuroblastoma Cells

Neuroblastoma(NB) is one of the common pediatric neoplasms and is derived from the sympathoadrenal lineage of the neural crest. Prognostic variability partly depends upon patient age at diagnosis and tumor stage. One intriguing feature of NB is that some disseminated tumors in infants may regress spontaneously even without treatment. Numerous investigations have sought tumor markers that are correlated with tumor stage and prognosis. Trks proto-oncogene expression has recently been found to play an important role in regulating the biology of NB.The family of homologous neurotrophins(NTs) includes nerve growth factor (NGF) , brain-neurotrophic factor(BDNF) , neurotrophin-3 ( NT-3) and neuro-trophin-4/5 ( NT-4/5). Recently, the three Trk genes for the neurotrophic family have been cloned. The genes TrkA, TrkB and TrkC encode the primary receptors for NGF, BDNF and NT-3, respectively. The cellular effect of NTs are mediated by binding and activating transmembrane Trks. There is considerable interest in the role of the Trks family of neurotrophin receptors in regulating the survival, growth and differentiation of NB cells. The exploration of Trk kinase inhibitors as an effective treatment for NB are stemmed from the premise that NB cell growth utilizes a NT/Trk signal transduction pathway for continued cell survival and proliferation.To evaluate the clinical significance of expression of Trk genes in NB, we studied the relationship between tumor differentiation, stage, prognosis and the expression of these receptors in NB tissue samples by SABC immunohistochemis-try and reverse transcription-polymerase chain reaction respectively. We thinkthat clinic laboratories in general hospitals easily could adopt these methods to assist with the molecular and genetic evaluation of patients with NB. We also investigate the effect of K252a on the morphology and growth of SY5 Y cells and its mechanisms of action in vitro. These studies establish a good beginning for the anti-tumor efficacy of K252a analog in NB models.Methods1. Clinical Section(1) Clinical Materials. We studied clinical materials from 34 patients diagnosed for NB, 2 cases of ganglioneuroblastoma, or 4 cases of ganglioneuroma in our hospital from 1997 to 2003. All diagnoses of patients were confirmed by his-tologic assessment of tumor specimens obtained at surgery. The age of patients at diagnosis was ranged from 2 months to 14 years and the mean age was 3. 2 years. According to the criteria of Evans, all patients were divided into stage I (5), stageII (6), stage IVS (3), stage I (7) and stage IV(13).(2) Immunohistochemistry. The expression of Trk receptors was examined by SABC immunohistochemistry technique in 40 cases of NB. A semiquantitative scale to degree of Trk immunoreactivity was used in all NB specimens. A scale from 0 to 5 was used: 0, no staining; 1, trace immunoreactivity evident; 2, occasional cells stain; 3, minority of cells stain; 4, majority of cells stain; and 5, all cells stain intensely. 10 randomly selected regions of each specimen were examined under high power, each region was rated, and the median rating of the 10 regions was determined.(3 ) Reverse transcription-polymerase chain reaction ( RT-PCR ). The expression of Trk genes mRNA was semiquantitatively detected by RT-PCR technique in 27 cases of NB. Trk expression was semi quantified by densitometry normalised to (3-actin. According to the ratio difference, we classified these rates to the three scales: < 0. 5, no expression; 0. 5 -1.0, k?w expression; > 1. 0, high expression.2. Basal Section(l)Cell Cultures. The SY5Y human neuroblastoma cells were maintainedin an atomosphere of 5%C02in RPMI -1640.(2)Transfection of TrkA. The full-length TrkA was subcloned into the pB-STR1 vector and transfected into SY5Y cells using Iipofectamine. We classified these cells to the three groups; SY5Y group, SY5Y-Vec group and SY5Y-TrkA group.(3) TrkA mRNA Expression. The expression level of TrkA mRNA was seraiquantitatively detected by RT-PCR technique. The TrkA index was calculated as the ratio of the optical densities of the TrkA band to the (3-actin band, and it was used as an estimated of TrkA mRNA expression.(4) Morphological Changes. After a week in cell culture of the three groups, these cells was fixed with 1. 25% glutaraldehyde and Annexin-V staining. Morphological changes of the three groups was observed under the fluorescent microscope.( 5 ) Cell Survival Analysis. Cells were seeded into a 96-well plate at a density of 3 x 104cells/well. Six days after cell culture, the number of living cells was determined using conversion of MTT according to stand protocols. A multi-well scanner was used to measure the absorbance at 550 nM dual wavelengths. The DMSO treated control group was assigned a proliferation rate of 100%. Cell Proliferation Rate( % ) = Test Data / Control Data x 100%.Results1. Clinical Section(1 ) TrkA Expression and differentiation maturation. In NB lobule with more organized histology, a specific pattern of TrkA and TrkC immunoreactivity was in lower stage tumors more intense than in higher stage tumors. There was a strongly increasing tendency to the center of the lobule in TrkA and TrkC immunoreactivity and maturation. All patients of ganglioneuroblastoma(2) and gan-glioneuroma(4) intensely expressed TrkA and TrkC. Immunoreactivity of neuro-blastic tumor cells proper was confirmed in only 4 patients of NB.(2) Immunochemical staining grade for Trk expression. TrkA expression had a significant difference between survivor group and non survivor group ( P <0.01) and had also a significant difference between higher stage group and lower stage group(P <0.01) , but TrkB or TrkC expression had no significance among their groups (P > 0.05). With increasing tumor stage in 34 patients, immunore-activity of TrkA expression showed a gradually decreaseing trend.(3)Trk expression rates. The high and total rates of TrkA were expressed in significantly more lower stage group than higher stage group (P <0. 05) and the high level of TrkA expression was correlated positively with the two-year cumulative-survival rate of these patients( P <0. 01). The high and total rates of TrkB were expressed in significantly more higher stage group than lower stage group( P <0. 05 ) and the three rates of BDNF expression in the two groups wer-ent a statistical difference(P >0. 05) , but the co-expression ratio of TrkB and BDNF showed a remarkable significance in higher stage group more than lower stage group( P <0.05 ). Expression of TrkC was usually accompanied by expression of TrkA and the co-expression ratio of them had a statistical significance in lower stage group more than higher stage group (P <0.05) , but there was only a non-significant trend between TrkC and TrkA expression.2. Basal Section(1) K252a affects morphology of SY5Y cells. DMSO-treated SY5 Y cells retained a flat cell body morphology and didnt form aggregates. K252a induced SY5Y and SY5Y-Vec cells enlarging, clustering and long neuritic extensions, but K252a induced SY5Y-TrkA cells scattering, decreasing and warping.(2)K252a affects proliferation of SY5Y cells. The DMSO treated control group was assigned a prolieration rate of 100% whereas K252a treated other three groups had proliferation rates (% ) : SY5Y( 127 ± 14) ,SY5Y-Vec (123 ± 15) and SY5Y-TrkA (65 ±8). There was no significant difference between SY5Y group and SY5Y-Vec group, but there was a statistical significance among the other groups( P < 0. 01).Conclusions1. Trk receptor expression and NB cell differentiation was examined by im-munohistochemistry technique in NB tissues.2. The expession of Trk genes was semiquantitatively detected by RT-PCR technique in NB tissues.3. Trk gene expression can have important clinical significance of tumor stage and outcome for patients with neuroblastomas.4. K252a can promote the growth and differentiation of SY5Y cells without TrkA expression.5. K252a can inhibit the growth and differentiation of SY5Y cells with TrkA expression.