Isolation of immortalized, INK4a/ARF-deficient cells from the subventricular zone after in utero N-ethyl-N-nitrosourea exposure.
OBJECTIVE
Brain tumors, including gliomas, develop several months after rats are exposed in utero to N-ethyl-N-nitroso-urea (ENU). Although pathological changes cannot be detected until these animals are several weeks old, the process that eventually leads to glioma formation must begin soon after exposure given the rapid clearance of the carcinogen and the observation that transformation of brain cells isolated soon after exposure occasionally occurs. This model can therefore potentially provide useful insights about the early events that precede overt glioma formation. The authors hypothesized that future glioma cells arise from stem/progenitor cells residing in or near the subventricular zone (SVZ) of the brain.


METHODS
Cells obtained from the SVZ or corpus striatum in ENU-exposed and control rats were cultured in an epidermal growth factor (EGF)-containing, chemically defined medium. Usually, rat SVZ cells cultured in this manner (neurospheres) are nestin-positive, undifferentiated, and EGF-dependent and undergo cell senescence. Consistent with these prior observations, control SVZ cells undergo senescence by the 12th to 15th doubling (20 of 20 cultures). In contrast, three of 15 cultures of cells derived from the SVZs of individual ENU-treated rats continue to proliferate for more than 60 cell passages. Each of these nestin-expressing immortalized cell lines harbored a common homozygous deletion spanning the INK4a/ARF locus and was unable to differentiate into neural lineages after exposure to specific in vitro stimuli. Nevertheless, unlike the rat C6 glioma cell line, these immortalized cell lines demonstrate EGF dependence and low clonogenicity in soft agar and did not form tumors after intracranial transplantation.


CONCLUSIONS
Data in this study indicated that immortalized cells may represent glioma precursors that reside in the area of the SVZ after ENU exposure that may serve as a reservoir for further genetic and epigenetic hits that could eventually result in a full glioma phenotype.
Savarese
Todd M
TM
Departments of Neurology, Neurosurgery, and Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Jang
Taichang
T
Low
Hoi Pang
HP
Salmonsen
Rebecca
R
Litofsky
N Scott
NS
Matuasevic
Zdenka
Z
Ross
Alonzo H
AH
Recht
Lawrence D
LD
D020727:ADP-Ribosylation Factors / Q000172:deficiency* / Q000187:drug effects*; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002552:Cerebral Ventricles / Q000187:drug effects* / Q000378:metabolism*; D003342:Corpus Striatum / Q000187:drug effects* / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000172:deficiency* / Q000187:drug effects* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004815:Epidermal Growth Factor / Q000378:metabolism; D005038:Ethylnitrosourea / Q000633:toxicity*; D005260:Female; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency* / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D008565:Membrane Proteins; D012333:RNA, Messenger; D025803:Tumor Suppressor Protein p14ARF; D004815:Epidermal Growth Factor; C097313:ADP-ribosylation factor related proteins; D020727:ADP-Ribosylation Factors; D005038:Ethylnitrosourea