Pankaj Taneja

Antimutagenic potential of curcumin on chromosomal aberrations in Wistar rats.

Curcumin, a yellow pigment commonly used as a spice and food coloring agent is obtained from rhizomes of Curcuma longa and is a major chemopreventive component of turmeric. In the present set of investigations the antimutagenic potential of curcumin has been evaluated using in vivo chromosomal aberration assay in Wistar rats. Cyclophosphamide (CP), a well-known mutagen was given by intraperitoneal (i.p.) injection at the dose of 40 mg/kg body weight (b.w.). Curcumin was given at the dose of 100 and 200 mg/kg b.w. through gastric intubation for seven consecutive days prior to CP treatment. The animals were sacrificed at the sampling time of 24 h after treatment and their bone marrow tissue was analyzed for chromosomal damage and mitotic index. In CP treated animals a significant induction of chromosomal aberration was recorded with decrease in mitotic index. However, in curcumin-supplemented animals, no significant induction in chromosomal damage or change in mitotic index was recorded. In different curcumin-supplemented groups, a dose dependent significant decrease in CP induced clastogenicity was recorded. The incidence of aberrant cells was found to be reduced by both the doses of curcumin when compared to CP treated group. The anticytotoxic potential of curcumin towards CP was also evident as the status of mitotic index was found to show increment. The study revealed the antigenotoxic potential of curcumin against CP induced chromosomal mutations.

MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer

Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients.

Anticarcinogenic effect of black tea on pulmonary tumors in Swiss albino mice

The widespread consumption of tea as beverage throughout the world has stimulated interest in the possibility of its use in chemoprevention of cancer. The present set of investigation was carried out to evaluate the anticarcinogenic activity of black tea using lung tumorigenesis model in Swiss albino mice. Diethylnitrosoamine (DEN), a known inducer of pulmonary tumors was given at the multiple (total eight) doses of 20mg/kg body weight through oral intubation to Swiss albino mice. Simultaneously, three different groups of animals received 1, 2 and 4% aqueous black tea extracts (ATE) as a sole source of drinking solution. The positive control group received DEN treatment only. The animals were sacrificed after 28 weeks of the first dose of DEN. They were identified for different histological types of alveologenic pulmonary tumors. In the positive DEN treated group, higher incidences of pulmonary tumors were observed, while in ATE treated groups, a lower incidence of DEN induced lung tumorigenicity was recorded. The percentage of mice having lung tumors was decreased following ATE administration. Besides this, significant decrease in the number of tumors/mouse was observed in 2 and 4% ATE supplemented groups. The histological examination revealed a significant decrease in pulmonary adenomas at all doses of ATE. The number of animals showing pulmonary adenocarcinomas induced by DEN was found to be inhibited significantly by 4% ATE. In addition, splenomegaly was found to be protected by ATE administration.

Repression of Dmp1 and Arf transcription by anthracyclins: critical roles of the NF-κB subunit p65

Both genotoxic and oncogenic stress activates the nuclear factor-kappa B (NF-κB) and p53 proteins; however, the p53 activity is antagonized by NF-κB signaling. Dmp1 is a Myb-like transcription factor that activates the Arf-p53 pathway. The Dmp1 promoter was activated by a classical NF-κB activator tumor necrosis factor α, but repressed by treatment of cells with non-classical NF-κB activators, anthracyclins and UV-C. p65 and other subsets of NF-κB proteins were bound to the Dmp1 promoter following anthracyclin/UV-C treatment of rodent fibroblasts. This resulted in the downregulation of Dmp1 mRNA and protein. Repression of the Dmp1 transcription by anthracyclins depended on the unique NF-κB site on the promoter. Downregulation of p65 significantly attenuated the repression of the Dmp1 promoter by anthracyclins/UV-C. The amount of Dmp1 bound to the Arf promoter decreased significantly upon anthracyclin treatment; this, in turn, downregulated the Arf levels. Repression of the Arf promoter by p65 or anthracyclins depended on Dmp1, which was significantly attenuated in Dmp1−/− cells. Both Dmp1−/−and Arf−/−cells showed resistance to anthracyclin-induced cell death compared to wild-type cells; non-immortalized p65-knockdown cells were much more sensitive. Thus, the Dmp1-Arf pathway is repressed by p65 in response to genotoxic stress, which implicates a novel mechanism of p53 inactivation by NF-κB.

Expression of Dmp1 in specific differentiated, nonproliferating cells and its regulation by E2Fs

Dmp1 is a Myb-like transcription factor that transmits oncogenic Ras-Raf signaling to the Arf-p53 pathway and induces cell cycle arrest. Immunohistochemical staining was performed to identify the pattern of Dmp1 expression in normal murine tissues compared with the proliferation marker, Ki67. In thymus, the nuclei of mature T lymphocytes in the medulla were strongly positive for Dmp1, whereas Ki67 was detected only in the cortex. In intestine, Dmp1 was detected in the nuclei of superficial layers of the villi, whereas Ki67-positive cells were confined to the lower one-third of the crypt. Double staining for Dmp1 and Ki67 revealed that these two proteins were expressed in mutually exclusive fashion in nearly all the tissues examined. Subsets of E2Fs were specifically bound to the Dmp1 promoter upon mitogenic signaling and E2Fs 1-4 inhibited the Dmp1 promoter in a reporter assay. The Dmp1 promoter was repressed when the cells entered the S to G2/M phase of the cell cycle when both Dmp1 and Arf expressions were downregulated. The Dmp1 mRNA was not downregulated by serum in E2F-DB(+) cells, suggesting that the Dmp1 promoter repression is E2F-dependent. This explains why the Dmp1 and Ki67-positive cells are stained in mutually exclusive fashion in normal tissues.

The Arf-inducing transcription factor Dmp1 encodes a transcriptional activator of amphiregulin, thrombospondin-1, JunB and Egr1.

Dmp1 (Dmtf1) encodes a Myb‐like transcription factor implicated in tumor suppression through direct activation of the Arf‐p53 pathway. The human DMP1 gene is frequently deleted in non‐small cell lung cancers, especially those that retain wild‐type INK4a/ARF and/or p53. To identify novel genes that are regulated by Dmp1, transcriptional profiles of lung tissue from Dmp1‐null and wild‐type mice were generated using the GeneChip Microarray. Comparative analysis of gene expression changes between the two groups resulted in identification of numerous genes that may be regulated by Dmp1. Notably, amphiregulin (Areg), thrombospondin‐1 (Tsp‐1), JunB, Egr1, adrenomedullin (Adm), Bcl‐3 and methyl‐CpG binding domain protein 1 (Mbd1) were downregulated in the lungs from Dmp1‐null mice while Gas1 and Ect2 genes were upregulated. These target genes were chosen for further analyses since they are involved in cell proliferation, transcription, angiogenesis/metastasis, apoptosis, or DNA methylation, and thus could account for the tumor suppressor phenotype of Dmp1. Dmp1 directly bound to the genomic loci of Areg, Tsp‐1, JunB and Egr1. Significant upregulation or downregulation of the novel Dmp1 target genes was observed upon transient expression of Dmp1 in alveolar epithelial cells, an effect which was nullified by the inhibition of de novo mRNA synthesis. Interestingly, these genes and their protein products were significantly downregulated or upregulated in the lungs from Dmp1‐heterozygous mice as well. Identification of novel Dmp1 target genes not only provides insights into the effects of Dmp1 on global gene expression, but also sheds light on the mechanism of haploid insufficiency of Dmp1 in tumor suppression.

Transgenic and Knockout Mice Models to Reveal the Functions of Tumor Suppressor Genes

Cancer is caused by multiple genetic alterations leading to uncontrolled cell proliferation through multiple pathways. Malignant cells arise from a variety of genetic factors, such as mutations in tumor suppressor genes (TSGs) that are involved in regulating the cell cycle, apoptosis, or cell differentiation, or maintenance of genomic integrity. Tumor suppressor mouse models are the most frequently used animal models in cancer research. The anti-tumorigenic functions of TSGs, and their role in development and differentiation, and inhibition of oncogenes are discussed. In this review, we summarize some of the important transgenic and knockout mouse models for TSGs, including Rb, p53, Ink4a/Arf, Brca1/2, and their related genes.

Prognostic value of the hDMP1-ARF-Hdm2-p53 pathway in breast cancer

Our recent study showed critical roles of Dmp1 as a sensor of oncogenic Ras, HER2/neu signaling and activation of the Arf-p53 pathway. To elucidate the role of human DMP1 (hDMP1) in breast cancer, one hundred and ten pairs of human breast cancer specimen were studied for the alterations of the hDMP1-ARF-Hdm2-p53 pathway with follow up of clinical outcomes. Loss of heterozygosity (LOH) of the hDMP1 locus was found in 42% of human breast carcinomas, while that of INK4a/ARF and p53 were found in 20 and 34%, respectively. Hdm2 amplification was found in 13% of the same sample, which was found independently of LOH for hDMP1. Conversely, LOH for hDMP1 was found in mutually exclusive fashion with that of INK4a/ARF and p53, and was associated with low Ki67 index and diploid karyotype. Consistently, LOH for hDMP1 was associated with luminal A category and longer relapse-free survival, while that of p53 was associated with non-luminal A and shorter survival. Thus, loss of hDMP1 could define a new disease category associated with prognosis of breast cancer patients. Human breast epithelial cells/cancer cells with wild-type p53 were sensitive to growth inhibition by activated Dmp1:ER while those that delete p14ARF or p53, and/or Hdm2 amplification showed partial or nearly complete resistance, indicating that p53 is a critical target for hDMP1 to exhibit its biological activity.

Role of DMP1 and its future in lung cancer diagnostics.

Lung cancer is the most lethal carcinoma worldwide. Mutations of p53, inactivation of p16INK4a, and overexpression of cyclins E, A and B are independently associated with poor prognoses of patients, while the prognostic value of cyclin D1 or RB expression is inconclusive. Cyclin D binding myb-like protein 1 (Dmp1) encodes a DNA binding protein that receives signals from oncogenic Ras and functions as a tumor suppressor by activating the Arf–p53 pathway. Dmp1 has been shown to be haplo-insufficient for tumor suppression in mouse models including K-ras-mediated lung carcinogenesis. The human DMP1 gene is located on chromosome 7q21, and our recent results revealed that the hDMP1 gene is deleted, but not mutated or silenced, in approximately 40 % of human non-small-cell lung carcinomas. These cases typically retained wild-type ARF and p53 and expressed very low levels of the hDMP1 protein. Thus, hDMP1 loss could be a novel diagnostic marker for non-small-cell lung carcinomas.

Emerging roles of DMP1 in lung cancer

The Ras-activated transcription factor DMP1 can stimulate Arf transcription to promote p53-dependent cell arrest. One recent study deepens the pathophysiologic significance of this pathway in cancer, first, by identifying DMP1 losses in human lung cancers that lack ARF/p53 mutations, and second, by demonstrating that Dmp1 deletions in the mouse are sufficient to promote K-ras-induced lung tumorigenesis via mechanisms consistent with a disruption of Arf/p53 suppressor function. These findings prompt further investigations of the prognostic value of DMP1 alterations in human cancers and the oncogenic events that can cooperate with DMP1 inactivation to drive tumorigenesis.