Kim et al
the fraction of the neurite-positive cells was calcu-
lated. Cell counting was performed after trypan blue
staining. To assess the trkA specificity of the biologic
effects that followed NGF treatment, the NGF experi-
ments were repeated in the presence of 100 nM of
K-252a (Kamiya Biochemicals, Thousand Oaks, Cali-
fornia), which was applied 1 hour before NGF treat-
ment. The cells were also treated with 100 ng/ml of
BDNF, and its effects were analyzed.
Cavazzana AO, Ninfo V, Roberts V, Triche TJ (1992). Periph-
eral neuroepithelioma: a light microscopic, immunocyto-
chemical, and ultrastructural study. Mod Pathol 5:71–78.
Chan HSL, Gallie BL, DeBoer G, Haddad G, Ikegaki N,
Dimitroulakos J, Yeger H, Ling V (1997). MYCN protein
expression as a predictor of neuroblastoma prognosis. Clin
Cancer Res 3:1699–1706.
Furukawa K, Estus S, Fu W, Mark RJ, Mattson MP (1997).
Neuroprotective action of cycloheximide involves induction
of bcl-2 and antioxidant pathways. J Cell Biol 136:1137–
Immunoblotting
1149.
The changes in the levels of neuronal markers and
EWS-Fli-1 protein were measured by immunoblotting of
Kim CJ, Matsuo T, Lee K-H, Thiele CJ (1999). Up-regulation
of insulin-like growth factor-II expression is a feature of trkA
but not trkB activation in SH-SY5Y neuroblastoma cells.
Am J Pathol 155:1661–1670.
30 g of total protein lysates obtained from their respec-
tive experiments. The proteins were probed with rabbit
polyclonal antibody to bcl-2 (N-19; Santa Cruz Biotech-
nology, Inc.), mouse monoclonal anti-neurofilament an-
tibody (2F11; NeoMarkers, Fremont, California), and rab-
bit polyclonal anti-Fli-1 antibody (C-19; Santa Cruz
Biotechnology, Inc.). The antibody to neurofilament used
recognizes the phosphorylated form of the 200-kd and
Klein R, Jing S, Nanduri V, O’Rourke E, Barbacid M (1991).
The trk proto-oncogene encodes a receptor for nerve growth
factor. Cell 65:189–197.
Labouyrie E, Dubus P, Groppi A, Mahon FX, Ferrer J, Parrens
M, Reiffers J, de Mascarel A, Merlio JP (1999). Expression of
neurotrophins and their receptors in human bone marrow.
Am J Pathol 154:405–415.
68-kd subunits of neurofilaments.
3
[
H]dthymidine Uptake Analysis
Ladanyi M, Heinemann FS, Huvos AG, Rao HP (1990). Neural
differentiation in small round cell tumors of bone and soft
tissue with the translocation t(11;22)(q24;q12). Hum Pathol
3
In a 96-well plate, 5ϫ10 cells were split in quadrupli-
cate. On the next day (day 0), NGF was added to the
individual wells at a concentration of 100 ng/ml. Cells
were cultured for 3 days (day 3) and 1 Ci of [ H]d-
thymidine (ICN, Costa Mesa, California) was applied to
the individual well. Cells were harvested 20 hours after
the [ H]dthymidine application, and radioactivity was
measured using a liquid scintillation counter. Every
experiment was repeated at least three times.
21:1245–1251.
3
Lamballe F, Klein R, Barbacid M (1991). TrkC, a new member
of the trk family of tyrosine kinases, is a receptor for
neurotrophin-3. Cell 66:967–979.
3
Linnoila RI, Tsokos M, Triche TJ, Marangos PJ, Chandra RS
(1986). Evidence for neural origin and PAS-positive variants
of the malignant small cell tumor of thoracopulmonary region
(“Askin tumor”). Am J Surg Pathol 10:124–133.
References
Lipinski M, Braham K, Philip I, Wiels J, Philip T (1987).
Neuroectoderm-associated antigens of Ewing’s sarcoma cell
lines. Cancer Res 47:183–137.
Ambros IM, Ambros PF, Strehl S, Kovar H, Gadner H,
Salzer-Kuntschik M (1991). MIC2 is a specific marker for
Ewing’s sarcoma and peripheral primitive neuroectodermal
tumors: evidence for a common histogenesis of Ewing’s
sarcoma and peripheral primitive neuroectodermal tumors
from MIC2 expression and specific chromosome aberration.
Cancer 67:1886–1893.
Llombart-Bosch A, Contesso G, Peydro-Olaya A (1996).
Histology, immuno-histochemistry, and electron microscopy
of small round cell tumors of bone. Semin Diagn Pathol
13:153–170.
Matsushima H, Bogenmann E (1993). Expression of trkA
cDNA in neuroblastomas mediates differentiation in vitro and
in vivo. Mol Cell Biol 13:7447–7456.
Askin FB, Rosai J, Sibley RK, Dehner LP, McCalister WH
(1979). Malignant small round cell tumor of the thoracopul-
monary region in childhood: a distinctive clinicopathologic
entity of uncertain histogenesis. Cancer 43:2438–2451.
McGregor LM, McCune BK, Graff JR, McDowell PR, Romans
KE, Yancopoulos GD, Ball DW, Baylin SB, Nelkin BD (1999).
Roles of trk family neurotrophin receptors in medullary thy-
roid carcinoma development and progression. Proc Natl
Acad Sci USA 96:4540–4545.
Azar CG, Scavarda NJ, Reynolds CP, Brodeur GM (1990).
Multiple defects of the nerve growth factor receptor in human
neuroblastomas. Cell Growth Differ 1:418–421.
Bhagirath T, Abe S, Nojima T, Yoshida MC (1995). Molecular
analysis of a t(11;22) translocation junction in a case of
Ewing’s sarcoma. Genes Chromosomes Cancer 13:126–
McKeon C, Thiele CJ, Ross RA, Kwan M, Triche TJ, Miser JS,
Israel MA (1988). Indistinguishable patterns of protoonco-
gene expression in two distinct but closely related tumors:
Ewing’s sarcoma and neuroepithelioma. Cancer Res 48:
132.
Brodeur GM, Nakagawara A, Yamashiro DJ, Ikegaki N, Liu
XG, Azar CG, Lee CP, Evans AE (1997). Expression of trkA,
trkB and trkC in human neuroblastomas. J Neurooncol
4307–4311.
Miknyoczki SJ, Land D, Huang L, Klein-Szanto AJ, Dionne
CA, Ruggeri BA (1999). Neurotrophins and trk receptors in
human pancreatic ductal adenocarcinoma: expression pat-
terns and effects on in vitro invasive behavior. Int J Cancer
81:413–427.
31:49–55.
Cavazzana AO, Miser JS, Jefferson J, Triche TJ (1987).
Experimental evidence for a neural origin of Ewing’s sarcoma
of bone. Am J Pathol 127:507–518.
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28 Laboratory Investigation • February 2002 • Volume 82 • Number 2