Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene.

Definitive information about the number and nature of discrete steps of tumorigenesis is enigmatic. To understand the multistep nature of carcinogenesis, an in vitro model of 20-Methylcholanthrene-treated primary fibroblast cells CNCI-PM-20, from 20-day old Swiss mouse embryo was used. Visible neoplastic changes with distinct morphological variations along with specific chromosomal aberrations like Robertsonian metacentrics, double and single-minute chromosomes and aneuploidy were observed from Passage-20 onwards. The cell cycle profile showed gradual increase in G(2)/M population till P-32, followed by evasion of block from P-36 onwards. Gradual increase in expression of C-myc, CyclinD1 and a decrease in expression of P21 was observed from P-20 onwards. CDC25A expression was significantly increased at P-27 and remained more or less constant in subsequent passages. Additionally, an increased P16 and P53 expression were seen at P-20 followed by their significant down-regulation at P-32. An increased level of phosphorylated retinoblastoma (ppRb) was observed from P-27, probably responsible for a compromised G(1)/S checkpoint. The inactivation of p21 and p16 might be due to their promoter hyper-methylation as suggested through de-methylation experiment by 5-aza-deoxycytidine at P-42. G(2)/M checkpoint abrogation was marked by gradual increase in expression of CyclinB1 and Cdc20, and a significant increase of Mad2 at P-20. Interestingly, increased expression of phospho-ATM, ATR and phospho-Chk1 were also seen at P-20 followed by their down-regulation at subsequent passages, indicating a perturbation of DNA damage response pathway at early passages. Our findings therefore dramatize the multiple genetic events that can cooperate to promote tumorigenesis.

Differential alterations in metabolic pattern of the spliceosomal uridylic acid-rich small nuclear RNAs (UsnRNAs) during malignant transformation of 20-methylcholanthrene-induced mouse CNCI-PM-20 embryonic fibroblasts.

Differential alterations of the spliceosomal Uridylic acid rich small nuclear RNAs (UsnRNAs) (U1, U2, U4, U5, and U6) are reported to be associated with cellular proliferation and development, but definitive information is scarce and also elusive. An attempt is made in this study to analyze the metabolic patterns of major spliceosomal UsnRNAs, during tumor development, in an in vitro carcinogenesis model of 20-methylcholanthrene (MCA)-transformed Swiss Mouse Embryonic Fibroblast (MEF), designated as CNCI-PM-20. MEF cells, after treatment with 20-MCA, progressed through a sequence of passages with distinct and heritable changes, finally becoming neoplastic at passage-42 (P42). A differential expression pattern of major UsnRNAs was observed during this process. The abundance of U1 was 20% below control (P1) at passage-20 (P20), followed by a gradual increase up until P42 (approximately 12% above the P1 value). The abundance of U2 was more or less constant during the cellular transformation. U4 showed a trend of increase, with above 30% abundance than control at P20, followed by a significant increase at P36 and P42 (1.5- and 2-fold, respectively, P-value <0.01). U5 also followed an identical pattern, with an increase of 70% compared to control (P-value <0.05) at P42. Interestingly, U6 gradually decreased from P20 onwards up until P42, with 22% at P20 and 67% at P42 (P-value <0.01). An overall significant quantitative alteration in abundance of U4, U5, and U6, observed in our study, contributes to the understanding of the fact that, the metabolism of major spliceosomal UsnRNAs is differentially regulated during the process of neoplastic transformation.

A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo.

BACKGROUND: In search of a suitable GSH-depleting agent, a novel copper complex viz., copper N-(2-hydroxyacetophenone) glycinate (CuNG) has been synthesized, which was initially found to be a potential resistance modifying agent and later found to be an immunomodulator in mice model in different doses. The objective of the present work was to decipher the effect of CuNG on reactive oxygen species (ROS) generation and antioxidant enzymes in normal and doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing Swiss albino mice. METHODS: The effect of CuNG has been studied on ROS generation, multidrug resistance-associated protein1 (MRP1) expression and on activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). RESULTS: CuNG increased ROS generation and reduced MRP1 expression in EAC/Dox cells while only temporarily depleted glutathione (GSH) within 2 h in heart, kidney, liver and lung of EAC/Dox bearing mice, which were restored within 24 h. The level of liver Cu was observed to be inversely proportional to the level of GSH. Moreover, CuNG modulated SOD, CAT and GPx in different organs and thereby reduced oxidative stress. Thus nontoxic dose of CuNG may be utilized to reduce MRP1 expression and thus sensitize EAC/Dox cells to standard chemotherapy. Moreover, CuNG modulated SOD, CAT and and GPx activities to reduce oxidative stress in some vital organs of EAC/Dox bearing mice. CuNG treatment also helped to recover liver and renal function in EAC/Dox bearing mice. CONCLUSION: Based on our studies, we conclude that CuNG may be a promising candidate to sensitize drug resistant cancers in the clinic.

Overcoming drug-resistant cancer by a newly developed copper chelate through host-protective cytokine-mediated apoptosis.

PURPOSE: Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines. EXPERIMENTAL DESIGN: Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing mice and doxorubicin-resistant sarcoma 180-bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay ex vivo. IFN-gamma and tumor necrosis factor-alpha were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-gamma and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-gamma and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells. RESULTS: CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-gamma and/or tumor necrosis factor-alpha, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-gamma-producing T cells in the ascetic tumor site. CONCLUSION: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.