Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.
BACKGROUND
Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.


RESULTS
Distinct cytoplasmic rods (∼3-10 µm in length) and rings (∼2-5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.


CONCLUSIONS
RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
Department of Oral Biology, University of Florida, Gainesville, Florida, United States of America.
Wendy C
WC
Carcamo
Minoru
M
Satoh
Hideko
H
Kasahara
Naohiro
N
Terada
Takashi
T
Hamazaki
Jason Y F
JY
Chan
Bing
B
Yao
Stephanie
S
Tamayo
Giovanni
G
Covini
Carlos A
CA
von Mühlen
Edward K L
EK
Chan
D000818:Animals; D053898:Biosynthetic Pathways / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D003570:Cytidine Triphosphate / Q000378:metabolism*; D022083:Cytoplasmic Structures / Q000187:drug effects / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D013997:Time Factors
D004791:Enzyme Inhibitors; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D007168:IMP Dehydrogenase; C501687:IMPDH2 protein, human