Department of Biological Sciences

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    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
    (Wiley, 2009-06-03) Mukherjee, Sudeshna
    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 (∼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.
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    Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene
    (Wiley, 2011-04-23) Mukherjee, Sudeshna
    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 G2/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 G1/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. G2/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