Aruna S. Jaiswal

β-Catenin-mediated transactivation and cell–cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells

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 G2/M phase arrest and apoptosis in HCT-116(p53+/+), HCT-116(p53−/−) and HCT-116(p21−/−) cell lines. We further investigated the association of the β-catenin-mediated c-Myc expression and the cell–cell adhesion pathways in curcumin-induced G2/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of β-catenin, decreased transactivation of β-catenin/Tcf-Lef, decreased promoter DNA binding activity of the β-catenin/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 G2/M phase arrest. The decreased transactivation of β-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of β-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell–cell adhesion proteins β-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 G2/M phase arrest and apoptosis in HCT-116 cells.

Genome based cell population heterogeneity promotes tumorigenicity: The evolutionary mechanism of cancer

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. J. Cell. Physiol. 219: 288–300, 2009.

Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro.

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–G2/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 re-established cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the soft-agar. 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.

Tumor Suppressor APC Blocks DNA Polymerase β-dependent Strand Displacement Synthesis during Long Patch but Not Short Patch Base Excision Repair and Increases Sensitivity to Methylmethane Sulfonate

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 β and blocks DNA polymerase β-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.

Upstream stimulating factor‐1 (USF1) and USF2 bind to and activate the promoter of the adenomatous polyposis coli (APC) tumor suppressor gene

The adenomatous polyposis coli (APC) gene product is involved in cell cycle arrest and apoptosis, and loss of function is associated with the development of colorectal carcinogenesis. Although it has been demonstrated that the APC gene is inducible, its transcriptional regulation has not been elucidated. Therefore, we characterized the promoter region of the APC gene and transcription factors required for basal expression. The APC gene has a TATA‐less promoter and contains consensus binding sites for Octamer, AP2, Sp1, a CAAT‐box, and three nucleotide sequences for E‐box A, B, and M. The E‐boxes are functional in several cancer cell lines and upstream stimulating factor‐1 (USF1) and USF2 interact with these sites, with a preferred sequence‐specificity for the B site. Analysis of activation of the cloned APC promoter by USF1 and USF2 in transient transfection assays in HCT‐116 cells demonstrated that mutation of the E‐box B site completely abolished the basal promoter activity. Further, the ectopic USF1 and USF2 expression in HCT‐116 cells with deletion mutations of E‐box A, B, and M sites showed that these E‐boxes contribute to USF1‐ and USF2‐mediated transcriptional activation of the APC promoter, with maximum promoter activity being associated with the E‐box B site. Thus, USF1 and USF2 transcription factors are critical for APC gene expression. J. Cell. Biochem. 80:262–277, 2001.

p53-dependent Transcriptional Regulation of the APC Promoter in Colon Cancer Cells Treated with DNA Alkylating Agents*

The APC(adenomatous polyposis coli) gene product is involved in cell cycle arrest and in apoptosis. The loss of APC function is associated with the development of colorectal carcinogenesis. In previous studies, we have shown that theAPC gene is inducible and that the DNA damage-induced level of APC mRNA requires p53. In the present study, we examined the role of p53 in the transcriptional regulation ofAPC promoter and characterized two p53-binding sites on the cloned APC promoter (pAPCP). Results of electrophoretic mobility shift assay showed specific interactions of p53 protein with p53-binding site oligonucleotides. The DNA-protein complex formed in electrophoretic mobility shift assay was competed with unlabeled excess of p53-binding site oligonucleotide, unaffected with p53-binding site mutant or Sp1-binding site oligonucleotides, and supershifted with anti-p53 antibodies. In a transient transfection assay, the pAPCP promoter activity was lower in HCT-116(p53+/+) cellsversus HCT-116(p53−/−) cells. p53-dependent down-regulation was further confirmed after co-transfection of pAPCP plasmid with pCMV-p53 into HCT-116(p53−/−) and SAOS-2 (p53-negative) cells. However, the treatment of cells with DNA alkylating agents methylmethane sulfonate andN-methyl-N′-nitro-N-nitrosoguanidine, which cause phosphorylation of p53 at Ser15 and Ser392, induced pAPCP promoter activity in HCT-116(p53+/+) cells. Other than p53-binding sites, using deletion mutation constructs, we have shown thatN-methyl-N′-nitro-N-nitrosoguanidine-induced transcriptional activation of the pAPCP promoter in HCT-116(p53+/+) cells depended upon the Sp1-binding site and the E-box B site. From these results, we conclude that unphosphorylated p53 can down-regulate and phosphorylated p53 can up-regulate the pAPCP promoter activity involving the p53, Sp1, or E-box B elements. These studies are important to understanding the role of p53 and APC in DNA damage-induced cell cycle arrest and/or apoptosis of cancer cells.

A novel function of adenomatous polyposis coli (APC) in regulating DNA repair.

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 beta (Pol-beta) 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.

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polβ) or DNA polymerase β (polβ) gene-knockout (MEFpolβKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G2/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polβ-dependent pathway and by blocking both FEN1 and DNA ligase activity in polβ-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polβ-dependent and -independent LP-BER pathways in MEF cells.

Zinc stabilizes adenomatous polyposis coli (APC) protein levels and induces cell cycle arrest in colon cancer cells

In the present study, we investigated the mechanisms by which zinc causes growth arrest in colon cancer cells. The results suggest that zinc treatment stabilizes the levels of the wild‐type adenomatous polyposis coli (APC) protein at the post‐translational level since the APC mRNA levels and the promoter activity of the APC gene were decreased in HCT‐116 cells (which express the wild‐type APC gene) after treatment with ZnCl2. Increased levels of wild‐type but not truncated APC proteins were required for the ZnCl2‐mediated G2/M phase arrest in different colon cancer cell lines. We further tested whether serum‐stimulation, which induces cell cycle arrest in the S phase, can relieve ZnCl2‐induced G2/M phase arrest of HCT‐116 cells. Results showed that in the HCT‐116 cells pretreated with ZnCl2, the serum‐stimulation neither changed the distribution of G2/M phase arrested cells nor the increased levels of APC protein. The G2/M phase arrest correlated with retarded growth of HCT‐116 cells. To further establish that wild‐type APC protein plays a role in ZnCl2‐induced G2/M arrest, we treated SW480 colon cancer cells that express truncated APC protein. We found that ZnCl2 treatment did not induce G2/M phase arrest in SW480 cells; however, the cell growth was retarded due to the loss of E‐cadherin and α‐tubulin levels. These results suggest that ZnCl2 inhibits the proliferation of colon cancer cells (which carry the wild‐type APC gene) through stabilization of the APC protein and cell cycle arrest in the G2/M phase. On the other hand, ZnCl2 inhibits the proliferation of colon cancer cells (which carry the mutant APC gene) by disrupting cellular attachment and microtubule stability.

Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells

Our previous studies have shown that treatment with cigarette smoke condensate (CSC) transforms normal breast epithelial cell line, MCF-10A. In the present study, the mechanism of CSC-induced transformation of breast epithelial cells was examined. We first determined whether benzo[a]pyrene (B[a]P)- and CSC-induced levels of APC are capable of inhibiting long-patch base excision repair (LP-BER) since our earlier studies had shown that an interaction of APC with DNA polymerase β (pol-β) blocks strand-displacement synthesis. With the use of a novel in vivo LP-BER assay, it was demonstrated that increased and decreased APC levels in different breast cancer cell lines were associated with a decrease or increase in LP-BER activity, respectively. The effect of APC on LP-BER in malignant and pre-malignant breast epithelial cell lines was produced by either overexpression or knockdown of APC. Furthermore, it was shown that the decreased LP-BER in B[a]P- or CSC-treated pre-malignant breast epithelial cells is associated with an increased level of APC and decreased cell growth. Our results suggest that the decreased growth allows cells to repair the damaged DNA before mitosis, and failure to repair damaged DNA has the potential to transform pre-malignant breast epithelial cells.