The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53-and p21-independent G 2 /M phase arrest and apoptosis in HCT-116(p53 +/+ ), HCT-116(p53 7/7 ) and HCT-116(p21 7/7 ) cell lines. We further investigated the association of the b-catenin-mediated c-Myc expression and the cell -cell adhesion pathways in curcumin-induced G 2 /M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of b-catenin, decreased transactivation of b-catenin/Tcf-Lef, decreased promoter DNA binding activity of the bcatenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G 2 /M phase arrest. The decreased transactivation of b-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of b-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell -cell adhesion proteins b-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell -cell adhesion pathways, resulting in G 2 /M phase arrest and apoptosis in HCT-116 cells.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

In the present investigation, we report a previously unsuspected function of the tumor suppressor protein, APC (adenomatous polyposis coli), in the regulation of base excision repair (BER). We identified a proliferating cell nuclear antigen-interacting protein-like box sequence in APC that binds DNA polymerase ␤ and blocks DNA polymerase ␤-mediated strand-displacement synthesis in long patch BER without affecting short patch BER. We further showed that the colon cancer cell line expressing the wild-type APC gene was more sensitive to a DNA-methylating agent due to decreased DNA repair by long patch BER than the cell line expressing the mutant APC gene lacking the proliferating cell nuclear antigen-interacting protein-like box. Experiments based on RNA interference showed that the wild-type APC gene expression is required for DNA methylation-induced sensitivity of colon cancer cells. Thus, APC may play a critical role in determining utilization of long versus short patch BER pathways and affect the susceptibility of colon cancer cells to carcinogenic and chemotherapeutic agents.

Cancer progression represents an evolutionary process where overall genome level changes reflect system instability and serve as a driving force for evolving new systems. To illustrate this principle it must be demonstrated that karyotypic heterogeneity (population diversity) directly contributes to tumorigenicity. Five well characterized in vitro tumor progression models representing various types of cancers were selected for such an analysis. The tumorigenicity of each model has been linked to different molecular pathways, and there is no common molecular mechanism shared among them. According to our hypothesis that genome level heterogeneity is a key to cancer evolution, we expect to reveal that the common link of tumorigenicity between these diverse models is elevated genome diversity. Spectral karyotyping (SKY) was used to compare the degree of karyotypic heterogeneity displayed in various sublines of these five models. The cell population diversity was determined by scoring type and frequencies of clonal and non-clonal chromosome aberrations (CCAs and NCCAs). The tumorigenicity of these models has been separately analyzed. As expected, the highest level of NCCAs was detected coupled with the strongest tumorigenicity among all models analyzed. The karyotypic heterogeneity of both benign hyperplastic lesions and premalignant dysplastic tissues were further analyzed to support this conclusion. This common link between elevated NCCAs and increased tumorigenicity suggests an evolutionary causative relationship between system instability, population diversity, and cancer evolution. This study reconciles the difference between evolutionary and molecular mechanisms of cancer and suggests that NCCAs can serve as a biomarker to monitor the probability of cancer progression.Increasing evidence illustrates that the somatic evolution of cancer is similar to natural evolution with system stability mediated genetic heterogeneity playing a key role (Nowell, 1976;Crespi and Summers, 2005;Heng et al., 2006bHeng et al., , 2008Maley et al., 2006; Heng, 2007a,b,c;Goymer, 2008). This concept offers an explanation to many seemingly contradictory NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript findings in the field including the recent unexpected failure to identify a handful of commonly shared cancer genes from initial attempts to sequence the cancer genome (Bielas et al., 2006;Heng, 2007a;Heng et al., 2006a;Greenman et al., 2007;Wood et al., 2007). An emerging genome-centric concept on cancer evolution states that overall genome level variation coupled with stochastic gene mutations serve as a driving force of cancer evolution by increasing the cell population diversity (Heng et al., 2006a,b,c). The importance of non-clonal chromosome aberrations (NCCAs) (both structural and numerical) and their dynamic interplay with clonal chromosome aberrations (CCAs) in the immortalization process has been recently demonstrated and supports the genome-centric concept of cancer evolution (Heng et al., 2004...

In the present study, we showed that a single-dose treatment of normal breast epithelial cell line, MCF10A, for 72 h with cigarette smoke condensate (CSC) resulted in a transformed phenotype. The anchorage-dependent growth of these cells was decreased due to increased cell cycle arrest in S-G 2 /M phase; however, the surviving cells developed resistance due to an increased Bcl-xL to Bax ratio. Levels of PCNA and gadd45 proteins -involved in DNA repair in response to genomic damage -were increased, suggesting that the cells were responding to CSC-induced genomic damage. The transformation of MCF10A cells was determined by their colony-forming efficiency in soft-agar in an anchorage-independent manner. CSC-treated MCF10A cells efficiently formed colonies in soft-agar. We then re-established cell lines from the soft-agar colonies and further examined the persistence of their transforming characteristics. The reestablished cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the softagar. An increased staining of neuropilin-1 (NRP-1) further showed a transformation characteristic of MCF10A cells treated with CSC. In summary, our results suggest that CSC is capable of transforming the MCF10A cells in vitro, supporting the role of cigarette smoking and increased risk for breast cancer.

Development of colon cancer is a multistep process frequently involving mutations in both theIntestinal cells are constantly exposed to DNA-damaging agents from dietary toxins (1, 2). The resulting DNA damage, if not efficiently repaired, may result in genomic instability, leading to malignant transformation. The development and progression of colon cancer is a multistep process in which growth control mechanisms are impaired progressively. Mutations of the adenomatous polyposis coli (APC) 1 tumor suppressor gene, the Ki-ras oncogene, the deleted in colorectal cancer (DCC) gene, the p53 gene, and DNA mismatch repair (MMR) genes play important roles at different stages of colorectal carcinogenesis (for review, see Ref. 3). Among these, mutation of the APC gene is an early event in familial adenomatous polyposis (4, 5) and sporadic colorectal cancers (3, 5-7). Apart from colorectal cancers, mutations in the APC gene also are associated with malignant brain tumors (Turcot's syndrome (8)). Although APC is expressed constitutively within normal colonic epithelium, little is known about how mutations of, or abnormal expression of, APC contribute to the development of colon cancer. Previous studies have indicated that the cellular levels of wild-type APC are critical to cytoskeletal integrity (9, 10), cellular adhesion (11), and Wingless/Wnt signaling (3,(12)(13)(14)(15)(16)(17). The phenotype of a cell expressing a mutated APC gene can be reverted by increased expression of the remaining wild-type APC allele and provide evidence that overexpression of the wild-type APC gene alone can suppress tumorigenicity (18,19). Thus, understanding the mechanisms by which APC gene expression can be induced at the molecular level is critical.The tumor suppressor gene p53 is also frequently mutated in colon cancer cells (3). The wild-type p53 protein is necessary for monitoring the G 1 checkpoint, sensing DNA damage, assembling the DNA repair machinery, modulating gene amplification, or activating apoptosis to remove damaged cells (20). p53 activates transcription from specific DNA-binding sites and represses transcription in a binding site-independent manner. Treatment of cells with DNA-damaging agents induces nuclear accumulation of p53, which trans-activates cell cycle-and apoptosis-related genes (20). In previous studies, the transient expression of the wild-type APC gene into mammalian cells has been shown to cause cell cycle arrest (21) and apoptosis (22). However, whether APC expression in cells treated with DNAdamaging agents is increased and whether p53 plays any role in the expression of the APC are currently unknown. In the present investigation, we demonstrate that APC expression is, in fact, strongly induced after treatment of cells with DNAdamaging agents, such as the potent colon carcinogen Nmethyl-NЈ-nitro-N-nitrosoguanidine (MNNG) (for review, see Ref. 23). Furthermore, results indicated that the increased expression of the APC after MNNG treatment is dependent upon p53 expression.Mammalian cells respond through a v...

Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase β (Pol-β) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis and chemotherapy by determining whether cells with DNA damage survive or undergo apoptosis. In this review, we summarize the evidence supporting this novel concept and suggest that these results will have implications for the development of more effective strategies for chemoprevention, prognosis, and chemotherapy of certain types of tumors.

The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase β (Pol-β) and identified a potent small molecular weight inhibitor, NSC-666715. We determined the specificity of this small molecular weight inhibitor for Pol-β by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single-nucleotide and long-patch BER. The binding specificity of NSC-666715 with Pol-β was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug temozolomide (TMZ) to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-β activity. It blocked Pol-β-directed single-nucleotide and long-patch BER without affecting the activity of APE1, Fen1, and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-β and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the "proof-of-concept" for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer.