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hamster embryo fibroblasts in culture. Bipasha Bosea ... evaluation. Earlier studies from our laboratory have ... causes transformation of Syrian hamster embryo.
Indian Journal of Experimental Biology Vol. 44, September 2006, pp. 693-698

Differential role of MAP kinase isoforms in malachite green transformed Syrian hamster embryo fibroblasts in culture Bipasha Bosea, Rekha R Gourb, Leena Motiwalea & K V K Raoa* a

Chemical Carcinogenesis Group and bImmunology Group, Khanolkar Shodhika, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India Received 5 January 2006; revised 25 May 2006 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. Keywords: Embryo cell, Increased S-phase, Malachite green, Malignant transformation, MAP kinases, Syrian hamster embryo cells.

Mitogen activated protein (MAP) kinases are a group of signaling proteins which mediate the transmission of extracellular signals/molecules to the intracellular targets. These are both tyrosine and serine/threonine phosphorylated enzymes which are activated by a wide variety of stimuli from the cell surface receptors to the nucleus. The three-tiered signaling cascade is highly conserved in mammalian system, which can be elucidated by upstream (from nucleus to cell surface receptor) or downstream (from cell surface receptor to the nucleus) manner1. The cascade is as follows: MAPKKK→MAPKK→MAPK2. Mammals express at least six distinctly regulated groups of MAP kinases. However, three of them are well characterized. These are extracellular regulated kinase (ERK), JUN-N-terminal kinase (JNK) and p38 kinase (α,β,γ). Activated MAP kinases are translocated into the nucleus. In mammals, a number of nuclear MAP kinase targets have been identified, including the ternary complex factors in the ETS (erythroblast transformation specific) family that stimulate the expression of the c-fos gene via serum response element (SRE). As per the classical concept, ERKs _________________ *Correspondent author Phone: 91-22-26240557 E-mail: [email protected]

group of MAP kinases were believed to be mainly associated with cell survival, proliferation, oncogenesis, and JNKs and p38 kinases with stress stimuli resulting in apoptosis. However, as per new findings, the activation of various MAP kinases isoforms depends on cell type and stimulus specificity. p38 kinases initially believed to be stress associated apoptosis inducer are involved in maintaining transformation in human breast cancer MCF-7 cell line through AP-1 (Activator Protein-1) activation3. Also the development of certain other cancer like diffused large B cell lymphoma (DLBCL), a form of high grade non-Hodgkin lymphoma, requires a consistent phosphoactivation of p38 MAP kinases which on inhibition results in apoptosis and thereby tumor regression4. Malachite green (MG), CAS no. 2437-29-8, chemical formula [(C23H25N2)(C2HO4)]2.C2H2O4 is a green coloured textile dye. It is a food adulterant/food colour being rampantly used in the third world countries including India despite the ban5. Toxicologically, MG has been classified under category CIII by joint FAO/WHO expert committee on food additives, which implies that the available toxicological data is inadequate for its safety evaluation. Earlier studies from our laboratory have shown that MG is extremely cytotoxic to mammalian

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cells in culture and generates free radicals6, 7. It also causes transformation of Syrian hamster embryo (SHE) cells in culture8, 9. In order to understand the role of MAP kinase signal transduction pathway during MG induced transformation of SHE fibroblasts in culture, we have studied the profile of different MAP kinase isoforms in control and MG transformed SHE cells. These malignantly transformed cells were further characterized for overexpression of cyclin D1 gene as compared to their controls. Materials and Methods Chemicals— Antibodies specific to the total form of ERK, JNK, p38 kinase and phosphoactive JNK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for the phosphoactive ERK was purchased from Promega (Madison, WI, USA) and phosphoactive p38 was purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies conjugated with horseradish peroxidase (HRPO) were purchased from Amersham Biosciences (UK). Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Corp. (NY, USA). Malachite green was purchased from Sigma Chemical Co. (St Louis, MO, USA). Cell and culture conditions— Primary SHE cultures were set up from 10-14 day gestation fetuses collected aseptically. Cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) and gentamycin (50μg/ml) and maintained at 37°C in 5% CO2 atmosphere. For secondary cultures, cells were transferred by trypsinisation with 0.25% trypsin. MG treatment— Stock MG solution was prepared fresh in sterile saline, passed through millipore membrane filter, diluted with saline and added to the culture dishes at appropriate concentrations. Control cultures received only sterile saline at the same volume usually less than 50μl and at this level had no effect on cell growth. Morphological transformation and establishment of immortal cell line— Transformation assays were carried out as per the method described by Reznikoff et al.10. Transformed foci were classified as type II and type III using the morphological criteria described by Reznikoff et al.10. Some of these MG transformed type III foci were taken out of the petri dishes by selective trypsinisation and were used for establishing an immortal cell line by regular passaging of the cells

at three-day interval. These immortal cell lines formed colonies in soft agar and induced tumors in nude mice11. Cells were maintained up to 150 passages at the time of this investigation and were used for all studies. Primary cultures at passage 1 were used as controls. Western blotting— Western blotting was carried out as per the method described earlier12. Briefly equal amounts of proteins were subjected to SDSPAGE (10%), transferred to nitrocellulose membrane, blocked overnight with BSA (5%) followed by probing with respective antibodies. All the antibodies against the total forms of ERK, JNK and p38 kinase were used at a dilution of 1:1000 and the antibodies for phospho ERK, JNK and p38 kinase were used at a dilution of 1:6000, 1:1000 and 1:400, respectively. This was followed by the treatment with ECL 1 and 2. The blot was developed for 1-5min and then exposed to X-ray films in the dark. The blots were quantitated by densitometric scanning. Indirect immunofluorescence—Immunofluorescence was carried out as per the method described earlier12. Dual parameter flow cytometric analysis—Dual parameter flow cytometric analyses of the cells were done to measure the cell cycle specific DNA content in relation to the various intracellular selected antigens as per the protocol of Theron and Bohm13. Cells were fixed in ethanol (70%)-PBS and centrifuged at 1000 rpm at 4°C for 5 min and permeabilised for 10 min on ice. Thereafter, the cells were incubated with respective primary antibodies at a dilution of 1:400 in BSA (1%)-PBS overnight at 4°C. Further, the cells were washed twice in ice cold PBS (1X) and incubated with 1:40 dilution of secondary antibody for 30 min at room temperature. Then the cells were incubated with propidium iodide (10 μg/ml) and RNase (100μg/ml) for 30 min at 37°C. About 10,000 events were acquired on BectonDickinson FACS-SCAN on dual parameter scale (FL2A and FL-1H) and the results were analyzed using cell quest software (FL-2A corresponds to DNA content on the X-axis and FL-1H to the antigen expression on the Y axis). RNA isolation and Northern blot hybridisation— Total RNA was extracted from control and transformed SHE cells as per the method of Chomczynski and Sacchi14. RNA samples were electrophoresed on formaldehyde (1.2%) denaturing agarose gels and transferred to nytran membrane by

BOSE et al.: ROLE OF MAP KINASE IN MG TRANSFORMED FIBROBLASTS

capillary transfer. The cyclin D1 cDNA was labeled with α32P dCTP by random primer labeling to a specific activity of 108 cpm/μg DNA. The RNA blots were hybridized with the labeled probe for 48 hr at 42°C with gentle shaking15. Then the blots were exposed to X-ray film for 5 days at -70°C and autoradiograms were developed. Results Expression profile of total forms of MAP kinase— ERK, JNK and p38 kinase showed similar levels of expression in both control and transformed SHE cells (Fig. 1A). Fig.1B represents the densitometric analysis of the expression profile of MAP kinase isoforms in control and transformed SHE cells. Expression profile of phosphoactive forms of MAP kinase—Decreased expression levels of phosphoactive ERK and JNK were observed in transformed SHE cells as compared to control (Fig. 1C). However, a 10 fold increased expression of

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phosphoactive p38 kinase was observed in transformed SHE cells as compared to control (Fig. 1C). Fig. 1D represents the densitometric analysis of the expression profile of phosphoactivated forms of ERKs, JNKs and p38 kinase in control and transformed SHE cells. Immunolocalisation of total and phosphoactive forms of MAP kinases—Similar amounts of cytoplasmic localization of total ERK was observed in both control and transformed SHE cells. Whereas phosphoactive ERK was localized more in nucleus in control SHE cells as compared to transformed SHE cells. Total JNK was expressed in both nucleus and cytoplasm and showed similar amounts of expression in control and transformed SHE cells. However phosphoactive JNK was localized more in nucleus of SHE cells of control as compared to transformed SHE cells. Similar amount of expression of nuclear membrane as well as cytoplasmic localization of total p38 kinase was observed in both control and

Fig. 1—(A)-Western blot analysis of total forms of ERK, JNK and p38 kinase in control and MG transformed SHE cells line; (B)Densitometric analysis of blots probed with total forms of ERK, JNK and p38 kinase in control and MG transformed SHE cell line; (C)Western blot analysis of phosphoactive forms of ERK, JNK and p38 kinase in control and MG transformed SHE cells line and (D)Densitometric analysis of blots probed with phosphoactive forms of ERK, JNK and p38 kinase in control and MG transformed SHE cell line.

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transformed SHE cells. Whereas, phosphoactive p38 was expressed predominantly in the nucleus and was expressed more in transformed SHE cells as compared to control. MAP kinases expression profile in relation to cell cycle phase—Almost equivalent expression of total ERK, JNK and p38 kinase was observed in both control and transformed SHE cells (Fig. 2). The intensity of dots at different channels in X-axis ie, 200 and 400 represent the expression of the enzyme in G0-G1 and G2/M, respectively. In between area represents the expression of the enzyme at S-phase. Transformed SHE cells showed a decreased expression of phosphoactive ERK and JNK as compared to control (Fig. 2). However, an increased expression of phosphoactive p38 kinase was observed in transformed SHE cells as compared to control (Fig. 2). Expression of cyclin D1 gene by Northern blot hybridisation—Two prominent transcripts of 4.8 and 1.9 kb of cyclin D1 gene was observed without any alteration in the levels in control and transformed SHE fibroblasts (data not shown). Discussion Transformation process involves several changes in the genomic expression. The process of transformation is associated with transduction of extracellular signals into cytoplasm and further downstream that influence cell behaviour ranging from quiescence in normal cells to motility, resistance to apoptosis and entry into active cell cycle16. The MAP kinase signaling module has been found to be associated with almost all of the cellular processes. One of the most explored functions of MAP kinase is the gene expression in response to extracellular stimuli17. The three most well characterized MAP kinase pathways are ERK, JNK and p38 kinase, all of which are responsible for transformation depending on cell type and stimulus specificity. The various profiles of MAP kinase activation are known to have different effects. Constitutively activated mutant MEK activates ERK 1 and 2, which elicits transformation of fibroblasts18. However, JNK2 isoform is preferentially required for epidermal growth factor induced transformation of human A549 lung carcinoma cells19. The kinases in the MAP kinase signaling pathway transmit the signal from extracellular surface to nucleus. The signaling circuits may be completed by phosphorylation of upstream

effectors by downstream kinases, resulting in modulation of signals. It is the phosphorylated form (activated molecule), which actually enters the nucleus. In this study, an increased expression of phosphoactive p38 was associated with persistent nuclear localization of phosphoactive p38 in transformed SHE cells. It might be possible that this activated p38 molecule was responsible for activating various transcription factors thereby maintaining transformation. The MAP kinase pathway is activated after a variety of cellular stimuli and has been found to regulate numerous cellular processes, particularly the cell cycle20. Therefore, we have studied the correlation between MAP kinase isoforms and their cell cycle phase by dual parameter flow cytometric analysis. We have also observed a decreased expression of phosphoactive ERK and JNK are associated with an increased expression of phoaphoactive p38 kinase during transformation of SHE cells by MG. Northern blot hybridization with cyclin D1 mRNA showed no change in the expression of cyclin D1 mRNA in transformed SHE cells as compared to their controls, thus suggested that during transformation of SHE cells by MG other mechanisms might be operative. Stimulation of p38 MAP kinase activity occurs following dual phosphorylation of Threonine 180 and Tyrosine 182 in the activation loop which causes a conformation change that exposes the enzyme active site21. Activated p38 MAP kinase appears to be very commonly expressed in a constitutive manner in a broad range of human cancers including non-small cell lung carcinoma (NSCLC)22, breast23 and colorectal cancers24. These studies support very well our studies wherein the p38 MAP kinase was activated several fold during MG induced SHE cell transformation. Furthermore, transformation of follicular lymphoma (FL), the most common form of low grade non-Hodgkin’s lymphoma to high grade diffuse large B cell lymphoma (DLBCL) has been found to be associated with phospho p38 MAP kinase expression4. SB 203580, an inhibitor of p38 MAPK specifically induces caspase 3 mediated apoptosis in FL derived cell line and inhibition of lymphoma growth in NOD-SCID mice. It also suggests the role of inhibitors of p38 MAP kinase pathway as potential candidates for therapeutic drugs in p38 over

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Fig. 2—Dual parameter flow cytometric analysis showing the expression of total and phosphoactive ERK, JNK and p38 kinase in control and MG transformed SHE cell line.

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expressing tumors4. The present study also 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 therapy25. Further studies are in progress from this point of view. Acknowledgement This work was supported in part by a CSIR Grant No. 37 (1141)/03/EMR-11, New Delhi, India to Dr. K V K Rao. We also thank Dr Sanjay Gupta for his help in the experiments. References 1 Seger R & Krebs E G, The MAPK signalling cascade, FASEB J, 9 (1995) 726. 2 Chang L & Karin M, Mammalian MAP kinase signalling cascades, Nature, 410 (2001) 37. 3 Pramanik R, Xiaomei Qi, Borowicz S, Choubey D, Schultz R M, Han J & Chen G, P38 isoforms have opposite effects of AP-1 dependent transcription through regulation of c-Jun, J Biol Chem, 278 (2003) 4831. 4 Elenitoba-Johnson K S J, Jenson S D, Abbott R T, Palais R A, Bohling S D, Lin Z, Tripp S, Shami P J, Wang L Y, Coupland R W, Buckstein R, Perez-Ordonez B, Perkins S L, Dube I D & Lim M S, Involvement of multiple signalling pathways in follicular lymphoma transformation: p38mitogen activated protein kinase as a target for therapy, Proc Natl Acad Sci USA, 100 (2003) 7259. 5 Bhat R V & Mathur P, Changing scenario of food colours in India, Curr Sci, 74 (1998) 198. 6 Panandiker A, Fernandes C & Rao K V K, The cytotoxic properties of malachite green are associated with the increased demethylase, arylhydrocarbon hydroxylase and lipid peroxidation in primary cultures of Syrian hamster embryo cells, Cancer Lett, 67 (1992) 93. 7 Panandiker A, Maru G B & Rao K V K, Dose response effect of malachite green on free radical formation, lipid peroxidation and DNA damage in Syrian hamster embryo cells and their modulation by antioxidants, Carcinogenesis, 15 (1994) 2445. 8 Mahudawala D M, Redkar A A, Wagh A, Gladstone B & Rao K V K, Malignant transformation of Syrian hamster embryo (SHE) cells in culture by malachite green: An agent of environmental importance, Indian J Exp Biol, 37 (1999) 904. 9 Mahudawala D M, Redkar A A & Rao K V K, The malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by Malachite Green: The transformation is associated with enhanced vimentin phosphorylation, PCNA expression and BrdU incorporation, Cell Mol Biol Lett, 5 (2000) 75. 10 Reznikoff C A, Bertram J S, Brankow D W & Heidelberger C, Quantitative and qualitative studies of chemical

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