Expression of Vascular Endothelial Growth Factor (VEGF) in Human Neuroblastomas




Apurva.A.Modi

Introduction

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen known to play a crucial role in angiogenesis associated with tumor growth, diabetic retinopathy and inflammatory disorders [1]. VEGF has been reported to be secreted by several tumors that include lung, thyroid, breast, gastrointestinal tract, kidney, bladder, ovary [2], haematopoietic [3] and intracranial tumors [4]. Three forms of VEGF are expressed in humans: VEGFA, VEGFB and VEGFC.
VEGFA is a homodimeric glycoprotein of molecular mass 45,000 kilo Daltons and its gene, which contains 8 exons, has been localized to chromosome 6p12. Four VEGFA species of 121, 165, 189, 206 amino acids are produced in human cells as a result of alternative splicing of the VEGF coding region. VEGFA also called the vascular permeability factor (VPF) controls permeability by interacting with two endothelial tyrosine kinase receptors, FLT-1 and KDR/FLK [5].
VEGFB, also called vascular endothelial growth factor related factor (VRF) is present on chromosome 11q13. It has two isoforms of 186 and 167 amino acids, which show homology to VEGFA and are co-expressed in many tissues. However, in situ hybridization has revealed that VEGFB is expressed predominantly in muscular tissues and that its expression can be detected at an early stage in embryonic development [6].
VEGFC was identified as a result of its binding affinity with receptor tyrosine kinase FLT-4 expressed mainly in lymphatic endothelia that does not bind VEGFA or VEGFB [7].
Marked vascularization is a hallmark of many neoplasms in the nervous system and VEGF has been investigated as a potent mediator of angiogenesis in many brain tumors. In highly vascular tissues such as glioblastomas, hemangioblastomas and meningiomas there is a significant correlation between VEGF expression and vascular endothelial proliferation whereas tumors such as ependymomas and neuroectodermal tumors do not show a significant correlation [8]. However, in human neuroblastomas, VEGF expression has been shown to effect angiogenesis as well as neuroblastoma cell growth directly [9].
In this study, five human neuroblastoma cell lines were evaluated by RT-PCR for the expression of VEGF. Sequencing of the RT-PCR product was then done to confirm the expression of VEGF and also to identify the type of VEGF associated with angiogenesis in these cell lines.

Figures


Figure 1-DNase treated RNA samples from the five neuroblastoma cell lines. Intense 28S and 18S bands with apparent mobilities of 2Kb and 0.9Kb respectively were seen in all the samples. LA1-55n (lane 1), SH-SY5Y (lane 2), SH-EP1 (lane 3), LA1-5s (lane 4) and BE(2)-C (lane 5).


Figure 2-Visualization with ethidium bromide of the electrophoretic pattern in 0.8% agarose gel of RT-PCR products obtained with specific primers for VEGF cDNA. RT-PCR products were obtained for LA1-55n (lane 1), SH-SY5Y (lane 2), LA1-5s (lane 4) and BE(2)-C (lane 5). No RT-PCR products were obtained for SH-EP1 (lane 3) and dH2O (lane 6), which was used as the negative control.


Figure 3-Visualization with ethidium bromide of the electrophoretic pattern in 0.8% agarose gel of RT-PCR products obtained with specific primers for b-Actin. RT-PCR products were obtained for LA1-55n (lane 1), SH-SY5Y (lane 2), SH-EP1 (lane 3), LA1-5s (lane 4) and BE(2)-C (lane 5). No RT-PCR product was obtained in lane 6 (negative control), which contained dH2O instead of cDNA template.


RESULTS
RNA was extracted from the nueroblastoma cell lines and DNase treated as described in materials and methods. It was seen that all the five DNased RNA samples showed sharp and intense 28S and 18S ribosomal bands with apparent mobilities of about 2Kb and 0.9Kb respectively (Figure 1). The RNA was thus confirmed not to be degraded and also free of DNA contamination.

RT-PCR was performed as described in materials and methods and the RT-PCR products obtained with specific primers for VEGF cDNA were run on 0.8%agarose gel. It was seen that the five neuroblastoma cell lines showed RT-PCR products, which were approximately 200 bp long (Figure 2). La1-5Sn cell line (Lane 1) showed an intense band, SH-SY5Y (Lane 2) and BE(2)-C (Lane 5) cell lines showed lighter bands, LA1-5S cell line (Lane 4) showed a faint band and SH-EP1 (Lane 3) showed no band at all. This confirmed the ability of VEGF expression in four of the five cell lines. Moreover, the negative control (Lane 1) where dH2O was added instead of the cDNA template revealed no product, showing the lack of contamination and non-specific amplification.
The positive control for the RT-PCR was amplification of actin with b-Actin specific primers used on the cDNA of each nueroblastoma cell line. It was seen that all the cDNA samples produced intense bands at 700 bp (Figure 3, Lanes 1-5). This showed that the samples had amplifiable DNA and lacked PCR inhibitors.

Four of the five neuroblastoma cell lines, which gave RT-PCR products, were sequenced as described in materials and methods. The LA1-5S RT-PCR product was not sequenced due to the absence of a band on a 0.8% agarose gel. It was seen that VEGF expressed by the four neuroblastoma cell lines (LA1-55N, SH-SY5Y, LA1-5S and BE(2)-C) showed 100% homology to the published VEGF gene sequence . Hence, it was confirmed that the 200Kb bands seen on the agarose gel were indeed VEGF cDNA produced by the neuroblastoma cell lines. Moreover they showed 100% homology only to VEGFA gene sequence and very little homology to the VEGFB and VEGFC gene sequences.

DISCUSSION

Neuroblastomas are the most common pediatric neoplasms. They arise from the adrenal medulla or other areas of the sympathetic nervous system. Tumor stage, patient age and biological variables such as histopathology and DNA content have been shown to have a clinically relevant prognosis value in this tumor [10]. More recently, Meitar et al. [11] demonstrated a correlation between angiogenesis and poor outcome in human neuroblastomas.
VEGF has been reported to be an important angiogenesis causing factor in many human tumors including neuroblastomas [8]. This study was undertaken to establish the role of VEGF in angiogenesis in different neuroblastoma cell lines. Here, using the RT-PCR technique it was demonstrated that VEGF mRNA was expressed in neuroblastoma tumors which arise from neuroblastic cells (LA1-55N, SH-SY5Y), from I type cells [BE(2)-C] which are highly malignant and also from S type (substrate adhesive) cells (LA1-5S). S type cells are stromal cells that are non- malignant [10], non-neuronal neural crest cells that develop into Schwann cells and melanocytes in the CNS [12]. SH-EP1, which is an S type cell tumor, did not show expression of VEGF and this could be explained as a +/- result.
Malignant solid tumors depend on neo-vascularization for their growth and for dissemination. Tumor cells produce angiogenic factors that directly or indirectly activate endothelial cells and stimulate them to develop into blood vessels that grow toward the tumors [10]. Since, blood vessels even in tumors, express very low amounts of VEGF [13], the present results suggest that neuroblastoma cells produced VEGF in vivo.
Neuroblastoma, a well-vascularized tumor, frequently spreads haematogenously [12]. It has been reported that LA1-55n amplifies N-myc, an oncogene responsible for metastasis in various tumors whereas SH-EP1, SH-SY5Y have been reported to have only one copy of N-myc [11]. Consistent with these reports, LA1-55n produced a band with the highest intensity, thus suggesting that metastatic neuroblastoma tumors express greater amounts of VEGF.

REFERENCES

1. Leung DW, Cachianes G, Kuang WJ, et al. (1989) Vascular endothelial growth factor is a secreted mitogen. Science 246:1306-1309
2. Ferrara N, Davis Smith T (1997) The biology of vascular endothelial growth factor. Endocrine Rev 18:4-25
3. Bellamy WT, Richter L, Frutiger Y, Grogan TM (1999) Expression of vascular endothelial growth factor and its receptors in haematopoietic malignancies. Cancer Res 59(3): 728-733
4. Peitsh T, Valter MM, Wolf HK, Von Deimling A, Huang HJ, Cavenee WK, Weistler OD (1997) Expression and distribution of vascular endothelial growth factor protein in human brain tumors. Acta Nueropathol (Berl) 93(2): 109-117
5. Tischer E, Mitchell R, Hartman T, et al. (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266:11947-11954
6. Grimmond S, Lagercrantz J, Drinkwater C, Silins G, Townson S, Pollock P, Gotley D, Carson E, Rakar S, Nordenskjold M, Ward L, Hayward N, Weber G (1996) Cloning and characterization of a novel human gene related to vascular endothelial growth factor. Genome Res 6:124-131
7. Joukov V, Pajusola K, Kaipainen A, Chilov D, Lantinen I, Kukk E, Saksela O, Kalkkinen N, Alitalo K (1996) A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBOJ 15:290-298
8. Berkman RA, Merrill MJ, Reinhold WC, Monacci WT, Saxena A, Clark WC, Robertson JT, Ali IU, Oldfield EH (1993) Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms. J Clin Invest 91(1): 153-159
9. Takano S, Yoshii Y, Kondo S, Suzuki H, Maruno T, Shirai S, Nose T (1996) Concentration of vascular endothelial growth factor in the serum and tumor tissue of brain tumor patients. Cancer Res 56(9): 2185-2190
10. Langer I, Vertongen P, Perret J, Fontaine J, Atassi G, Robberecht P (2000) Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in human nueroblastomas. Med Pedatr Oncol 6:386-393
11. Meitar D, Crawford SE, Rademaker AW, Cohn SL (1996) Tumor angiogenesis with metastatic disease, N-myc amplification, and poor outcome in human nueroblastoma. J Clin Oncol 14:405-414
12. Meister B, Grunebach F, Bautz F, Brugger W, Fink FM, Kanz C, Mohle R (1999) expression of vascular endothelial growth factor (VEGF) and its receptors in human nueroblastoma. Eur J Cancer 35(3): 445-449
13. Rossler J, Breit S, Havers W, Schweigerer L (1999) Vascular endothelial growth factor expression in human nueroblastoma; up-regulation by hypoxia. Int J Cancer 81(1): 113-117


Full Paper

Acknowledgments

I thank Dr. Berish Rubin for us of his laboratory and resources necessary to carry out this research. I also thank Dr. Robert Ross and Sharon Thomas for providing the cell lines required for my research. Finally, I thank Rocco Coli, Sabrina Volpi and Ira Daly for their extreme patience, timely help and useful suggestions throughout the period of the research.


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