ABSTRACT
Malachite green (MG) induces DNA damage and malignant transformation of Syrian hamster embryo (SHE) cells in primary culture. In the present study, we have studied the role of all the three isoforms of mitogen activated protein (MAP) kinases i.e. ERK (extracellular regulated kinase), JNK (JUN- N- terminal kinase) and p38 kinase during transformation of SHE cells by MG. The results showed that transformed cells were associated with a decreased expression of phosphoactive ERK and JNK and increased expression of p38 kinase as evident from the Western blot, immunofluorescence and flow cytometry studies. Also, a persistent nuclear localization of p38 kinase was observed in the transformed cells. The present study indicated that p38 kinase was present at higher levels and seemed to be associated with transformation, which suggested that inhibitors of p38 kinase could serve in general as potential agents for selective cancer therapy.
Subject(s)
Coloring Agents/toxicity , Extracellular Signal-Regulated MAP Kinases/biosynthesis , Fibroblasts/enzymology , JNK Mitogen-Activated Protein Kinases/biosynthesis , Rosaniline Dyes/toxicity , p38 Mitogen-Activated Protein Kinases/biosynthesis , Animals , Blotting, Northern , Blotting, Western , Cell Culture Techniques , Cell Cycle/drug effects , Cell Line, Transformed , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Cricetinae , Cyclin D1/genetics , Cytoplasm/drug effects , Cytoplasm/enzymology , Fibroblasts/cytology , Fibroblasts/drug effects , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression/drug effects , Isoenzymes , MesocricetusABSTRACT
BACKGROUND: Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure of human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. METHODS: Cell transformation assays were carried out as described in the literature. Western blotting and flow cytometry were carried out by standard methods. RESULTS: In this study, we have studied the role of all three isoforms of mitogen-activated protein (MAP) kinases, i.e. extracellular regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and p38 kinase in the MG-transformed SHE fibroblasts compared to controls. Our results showed that transformed cells were associated with decreased expression of ERKs and JNKs as evidenced by Western blotting studies. However, the p38 MAP kinase was found to be upregulated. Flow cytometric DNA histogram analysis indicated an increase in the expression of S phase cells in the transformed cell line as compared to their control counterparts. CONCLUSIONS: The present studies indicate that decreased phosphoactive ERKs and JNKs and increased phosphoactive p38 kinase are associated with increased S phase cells during transformation of SHE cells by MG.
Subject(s)
Fibroblasts/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Rosaniline Dyes/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line, Transformed , Cricetinae , Embryo, Mammalian/cytology , Fibroblasts/enzymology , Mesocricetus , Phosphorylation/drug effects , S Phase/drug effectsABSTRACT
Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.
