Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. 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 Desacetylnimbin 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. or does it associate with accessory /replication proteins? Although answers to these are unknown, evidence from our laboratory and others suggests that MGMT specifically interacts with many cellular proteins and may have other functions [, ]. For example, using MGMT-Sepharose affinity chromatography and tandem mass spectrometry, we showed the specific interaction of MGMT with a diverse group of proteins involved in DNA replication (ORC1, MCM helicases, PCNA, DNA pol ) and cell cycle progression (CDKs, p21cip1, and ubiquitin pathway components) . A similar study by another group confirmed the specific binding of MGMT with various regulatory proteins in human glioma cells . Recently, we showed a specific association and fine interplay between human MGMT and estrogen receptor- proteins and their co-degradation after tumor cell treatments with either O6-benzylguanine or fulvestrant, their respective inhibitors . A previous study implied that the alkylated (inactivated) human MGMT is a negative regulator of ER-mediated transcription following DNA alkylation damage . Previously, we reported briefly on MGMT binding with PCNA  and the presence of MGMT in p21cip1-PCNA complexes . Human PCNA is a homotrimeric protein that encircles the duplex DNA forming a ring-shaped clamp and functions as a processivity factor by tethering replicative DNA polymerases . PCNA also provides a molecular platform that facilitates the myriad protein-protein and proteinCDNA interactions that occur at the replication fork. Numerous PCNA-associated proteins such as the FEN1 nuclease, DNA cytosine methyltransferase, and topoisomerase II compete for binding to a common surface on PCNA . Many of these partners contain a highly conserved PCNA-binding motif, QXXhXXaa (where h is a hydrophobic, aa are aromatic and X is any amino acid), referred to as a PCNA interacting protein (PIP) box [, ]. Besides being an essential part of DNA replication machinery, PCNA also plays important roles in cell cycle regulation (primarily in S-phase), Mouse monoclonal antibody to L1CAM. The L1CAM gene, which is located in Xq28, is involved in three distinct conditions: 1) HSAS(hydrocephalus-stenosis of the aqueduct of Sylvius); 2) MASA (mental retardation, aphasia,shuffling gait, adductus thumbs); and 3) SPG1 (spastic paraplegia). The L1, neural cell adhesionmolecule (L1CAM) also plays an important role in axon growth, fasciculation, neural migrationand in mediating neuronal differentiation. Expression of L1 protein is restricted to tissues arisingfrom neuroectoderm translesion synthesis, long-patch base excision repair and recombination . All DNA repair pathways including nucleotide excision repair (XP-A, XP-G), mismatch repair (MSH2, MSH6), and PARP-1 are associated with PCNA in some way or another . While PCNA exists in free and chromatin-bound states, the abundance of cellular PCNA is strictly controlled by the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A, which is an avid binder and sequestrator of the former . p21cip1, which is activated by the p53 tumor suppressor, plays essential roles in the DNA damage response by inducing cell cycle arrest, direct inhibition of DNA replication and apoptotic regulation. p21 interferes with PCNA-dependent DNA polymerase activity, thereby inhibiting DNA replication, however, DNA repair processes dependent on PCNA appear to be largely unaffected [, ]. Additionally, PCNA interfaces with the cell cycle by forming PCNA-p21/CDK-cyclin quaternary complexes Desacetylnimbin with both positive and negative signaling roles in cell cycle progression. Furthermore, PCNA is an integral component of the regulated and timely destruction of proteins during the S-phase and facilitates the formation of pre-replication complexes for the next cell cycle [, ]. In this process, PCNA assists with the recognition of target proteins by CRL4Cdt2 ub-ligase [, ]. No information is available on the involvement of either PCNA or other cell cycle proteins in the regulation of the one-step direct reversal reaction performed by MGMT. Here we describe a detailed analysis of MGMT interaction with PCNA and p21cip1 in isogenic cancer cell lines and demonstrate a PCNA-dependent downregulation of MGMT protein in S-phase. Materials and Methods Cell Culture and Reagents All cell lines were obtained within 6 months of time and were authenticated by the original sources and investigators. The human glioblastoma cell lines, SF188 (Univ. of California Desacetylnimbin Brain Tumor Bank, San Francisco, CA), GBM10 (Dr. Jann Sarkaria, Mayo Clinic, Rochester, MN), and T98G (ATCC). Other cell lines, HT29 colon cancer,.