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615 RES 21
TRB2 AND SKP2 IN THE RETINOBLASTOMA PATH-
WAY
Xiaoliang L. Xu1,4, Renbing Jia1,5, Nengyi Zhou1, Xianqun Fan5, David H.
Abramson2, David Cobrinik ,4 and Suresh C. Jhanwar1 (xux2@mskcc.org)
1. Departments of Pathology; 2. Ophthalmic Oncology Service 3;
Department of Pediatrics
4. Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,
New York, USA; 5. Department of Ophthalmology, Shanghai Ninth
People’s Hospital, Shanghai Jiaotong University, Shanghai, P. R. China.
Purpose. We previously reported that TRB2 and SKP2 are essential
for retinoblastoma pathogenesis. In this study, we examine how TRB2
regulates SKP2.
Methods. We knocked down (KD) or overexpressed TRB1, TRB2, and
related genes in retinoblastoma and RB1 gene in colon cancer and
neuroblastoma to clarify the TRB and Rb signaling pathway.
Results. TRB2-KD promoted TRB1 activation, Emi1 inactivation, Cdh1
activation, and SKP2 degradation, resulting in S phase arrest. Conversely,
TRB1 KD promoted Emi1 activation, SKP2 stabilization, resulting in G2/M
block. TRB1 activated PP1-CDC25C-CDK1-PLK1 to phosphorylate and
inactivate Emi1, resulting in activation of Cdh1. TRB2 counteracted TRB1
and maintained PP2A mediated Emi1 dephosphorylation and activation.
The TRB knockout promoted, whereas TRB2 KO suppressed anterior
pituitary tumor formation in RB1+/- mice. Phospho-Rb binds to TRB2,
PP2A, and Emi1 to form a nuclear complex, named as S phase promoting
complex (SPC), which is essential for S-phase progression. RB1-KD in
neuroblastoma and colon cancer cells caused TRB1 activation, SPC
dissociation, Emi1 inactivation, Cdh1 activation, and SKP2 degradation,
resulting in S phase arrest.
Conclusions. G1-S and G2-M transitions exhibit a critical balance
regulated by TRB1 and TRB2, in which TRB2 promotes, whereas TRB1
suppresses G1/S transition. Following Rb mutation, TRB1 is activated,
resulting in SPC dissociation, Emi1 inactivation, cdh1 activation, and
SKP2 degradation preventing cell cycle reentry, which is a second
assurance for cell cycle control. Thus, high level TRB2 in cone precursors
counteracts TRB1 by maintaining SPC integrity, disrupting the doubleassurance,
and promoting cell cycle entry in retinoblastoma. The SPC
integrity may be an optimal therapeutic target for retinoblastoma.
Financial disclosure. Research and Development Funds of Department of Pathology, MSKCC.
The Fund for Ophthalmic Knowledge. Gerber Foundation.
1515 RES 22
NESTED RT-PCR DETERMINATION OF GD2 SYNTHASE
AS A MARKER FOR MINIMAL DISEASE IN RETINOBLAS-
TOMA IN THE CEREBRO-SPINAL FLUID (CSF)
Viviana Laurent, Claudia Sampor, Jorge Rossi, Mariano Gabri, Maria TG
de Davila, Daniel Alonso, Guillermo Chantada (gchantada@yahoo.com)
Hospital JP Garrahan, Universidad de Quilmes, Argentina
Purpose. The standard criteria for evaluation of the CSF in retinoblastoma
includes neuro-imaging and cerebrospinal fluid (CSF) cytology. We
hypothesized that RT-PCR-based techniques might increase the yield for
determining minimal dissemination and potentially guide CNS directed
therapy.
Methods. We evaluated the CSF of children with retinoblastoma who
presented IRSS stage 2-4 (n=14) and selected stage 1 with pathology risk
factors (n=8) at diagnosis, at 6 and 12 months and at relapse. Standard
evaluation included cell count and microscopical examination of the
RESEARCH DAY
Abstracts
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cytoentrifugate. Minimal disease evaluation included immunocytology
for GD2 ganglioside and RT-PCR followed by nested-PCR amplification
of a GD2 synthase mRNA fragment.
Results. Three children underwent treatment for CNS invasion (2 trilateral)
diagnosed by standard evaluation. PCR was negative in both trilateral
cases and it was positive in the remaining case who had a persistent
positivity despite the CSF cytology cleared after chemotherapy. She had
a fatal CNS relapse thereafter. Eleven children with stage 2 to 4a disease
had no CNS involvement at diagnosis by standard evaluation and PCR
was positive in 4 (all of them had massive optic nerve involvement and 1
had also bone marrow metastasis). Two of them had a CNS relapse and
the remaining 2 are in complete remission for 9 and 18 months (followup
PCR is negative) after intensive therapy. CNS relapse occurred in 4
children with negative or not evaluable PCR (CNS mass with normal CSF
in 3). All children with stage 1 had negative CSF evaluation by all techniques
at all times and did not experience CNS relapse.
Conclusions. RT-PCR-based assays for the evaluation of the CSF status
in children with retinoblastoma may improve the sensitivity of standard
techniques by identifying minimal disease.
Financial disclosure. Supported by a grant from the Agencia de Promocion Cientifica y
Tecnologica, Ministerio de Ciencia, Tecnologia e Innovacion Productiva, Argentina, Fund for
Ophthalmic Knowledge, NYC, USA and Fundacion Natali Dafne Flexer, Buenos Aires, Argentina
1824 RES 23
SUPER-SELECTIVE INTRA-OPHTHALMIC ARTERY CHE-
MOTHERAPY: RETINAL ENDOTHELIAL TOXICITY IN
PRE-CLINICAL MODELS
Matthew W. Wilson, MD, FACS1,2,3, J. Scott Williams, MD, PhD4, John S.
Jackson, DVM5, Fan Wang, PhD6, Clinton F. Stewart, PharmD6, Timothy
D. Mandrell, DVM4, Barrett G. Haik, MD, FACS1,2, Jena J. Steinle, PhD1
(mwilson5@uthsc.edu)
University of Tennessee Health Science Center, 1. Hamilton Eye Institute,
Department of Ophthalmology; 4. Department of Radiology and 5.
Department of Comparative Medicine, Memphis, Tennessee, USA
St Jude Children’s Research Hospital, 2. Department of Surgery, Division
of Ophthalmology; 3. Department of Pathology; 6. Department of
Pharmaceutical Sciences, Memphis, Tennessee, USA
Purpose. To report in vitro and in vivo pre-clinical modeling of superselective
intra-ophthalmic artery chemotherapy (SSIOAC).
Methods. Cultured human retinal endothelial cells were exposed to
increasing concentration of melphalan and carboplatin. Post-exposure
cell death, proliferation and migration were measured. Surviving
cells were studied using microarray and ELISA. Six adult male Rhesus
macaques were randomly assigned to treatment with either 5 mg/30 mL
melphalan or 30 mg/30 mL carboplatin. Each animal underwent three
separate SSIOAC procedures at three-week intervals. Digital retinal
images were obtained during each infusion. Intravenous fluorescein
angiography was performed immediately after each procedure.
Results. Highest concentration of melphalan (4mg/ml) and carboplatin
(1mM) caused a 5 fold increase in cell death at 24 hours (p