Elsevier

Neoplasia

Volume 20, Issue 4, April 2018, Pages 305-323
Neoplasia

S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

https://doi.org/10.1016/j.neo.2018.01.010Get rights and content
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Abstract

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

Abbreviations

MGMT
O6-methylguanine DNA methyltransferase
PCNA
proliferating nuclear antigen
His Tag
Hexahistidine tag
DAPI
4,6-Diamidino-2-phenylindole
rMGMT
recombinant MGMT protein
tet
tetracycline
IB
immunoblot
WB
western blot
LV
lenti-viral transfection
AS
asynchronous cells
CS0
G1/S synchronized cells before release for cycling
CS2, CS4 and CS8
synchronized cells 2, 4 and 8 hours after release into cycling
TMZ
temozolomide
BCNU
1,3-bis (2-chloroethyl)-1-nitrosourea
Conc
concentration
CRL4Cdt2
PCNA-dependent ubiquitin ligase
Ub
ubiquitin
IP
immunoprecipitation
PIP-box
PCNA interacting protein motif.

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