Journal of Cell and Tissue Research Vol. 8 (1) 1211-1224 (2008) ISSN: 0973- 0028 (Available online at www.tcrjournals.com)
Original Article
DIFFERENTIAL FUNCTIONAL MODULATION OF THE DEATH PROTEINS IN IN VIVO AND IN VITRO MICROENVIRONMENTS IN EXPERIMENTAL BRAIN TUMORS (GLIOMA) BHATTACHARJEE, M., GHOSH, A., ACHARYA, S ., SARKAR, P., CHAUDHURI, S. ? AND CHAUDHURI, SWAPNA Cellular and Molecular Immunology Lab, Department of Physiology, Dr. B.C .Roy Postgraduate Institute of Basic Medical Sciences, Institute of Postgraduate Medical Education and Research, 244B, A.J.C. Bose Road, Kolkata 700020, India. E. Mail:
[email protected] Received: October 3, 2007; Accepted: October 15, 2007 Abstract: Experiments in field of oncology mostly require an in vitro experimental setup. However, the in vitro tumor environment may not always simulate the in vivo milieu. The basic aim was thus to find out the differential modulation of the same proteins in the two environments. For the purpose, the most extensively studied proteins- “the death proteins” were chosen of which Fas/FasL belonged to the surface receptor group, while the cytosolic proteins of choice were the intrinsic death regulators- Bcl-2, Bax, p53, caspase-8, caspase9 and caspase-3. A distinct disparity in orientation of the surface receptors were observed with that of the cytosolic proteins when the two microenvironments were compared. The death receptor (in this case Fas/CD95) of freshly isolated (in vivo) glioma cells helps the cells to escape immune surveillance while the same receptors in the culture microenvironment primes it towards an apoptotic death. In contrast, the cytosolic death regulators (p53, Bcl-2, Bax ) of cultured glioma cells as compared to freshly isolated cells strongly stabilizes the cultured cells in favor of survival. The caspases also behaved differentially in the two microenvironments. The study clearly points out that the same proteins orients themselves in different functional patterns in two microenvironments even though the cells belonged to the same tumor type. Though it is known that the culture microenvironment does not always suffice the in vivo condition, the study is the first of its kind in analyzing, documenting and elucidating the functional disparity of the same proteins in two aforesaid microenvironments. Key word: In vivo, In vitro, Apoptosis, Glioma, Microenvironment
INTRODUCTION To deal with the investigations of the mechanisms involved in malignant transformation, essentially an in vitro setup, either a cell line or cell culture is needed. Multiple tumor cell lines such as LN-229 (malignant glioma cell line)[1]; LN18, LN229, LN308, LN443 (human astrocytoma cell lines) [2]; KMBC and Mz-Cha-1 (Human cholangiocarcinoma cell lines) [3]; K562 (erythroblastoid cell line) [4,5] and cell cultures obtained from culture collections ECACC (European collection of cell
culture) [6], mixed population cultures such as B958-marmoset cultures [6] derived from blood and mixed glial cell cultures, are extensively used for multifarious research findings. Even thou gh it is known t hat the in vivo microenvironment does not always simulate the in vitro microenvironment, still required molecular expression of different proteins are studied and analyzed based on the data form in vitro set up. The differential functional disparity is often overlooked. In the present course of investigation,
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J. Cell Tissue Research we tried to adjudge the differential expression of 5 key apoptotic proteins (p53, Bcl-2, Bax, FasFasL, Caspase-8, Caspase 9 and Caspase 3) in freshly isolated normal brain cells, freshly isolated (in vivo) glioma cells from tumor induced animals and cultured (in vitro) glioma cells. Cell death proteins were chosen as parameters as because worldwide research to inflict apoptosis in tumor cells has reached its peak in the present decade. Multiple approaches with cultured tumor cells and cell lines have been undertaken to study the role and mode of action of the cell death proteins ther eby over looking the differ ences in the modulation of the death proteins that might occur between the in vitro and in vivo microenvironment. As contrary to our beliefs that the two micr oenvir onment simulate each other, we observed a striking difference in the level of protein expression in the 2 set ups. Disparity was also observed in the cytosolic protein expression as compar ed t o the sur face r ecept or pr otein expression in the two microenvironments. Study, analysis and documentation of the differences in the functional orientation of the same proteins in two different microenvironment (in vivo and in vitro) is first of its kind giving us an insight into the fact that how in reality the scientific data obtained from in vitro will not always suffice the in vivo data. MATERIALS AND METHODS Animals: experiments: Healthy newborn Druckray rats, 2–3 days old of both sexes along with the mother were maintained in our Laboratory for the purpose of investigations. 6 animals in each group were weaned at 30 days of age and housed separately in isolated cages. All animals were fed autoclaved Hind Lever pellet and water ad libitum and housed in a room with ambient temperature of 22°C in 12 h light/darkness cycle. The animals were grouped in batches of 6 for each experimental group, which are as follows: (i) Normal control (N), (ii) 3–5 days old neonatal animals injected with ethyl nitrosourea (ENU) intraperitoneally (i.p.) which had full grown tumors after reaching the age of 5 months (E). Rats were examined daily and weighed weekly throughout the experimental period. Maintenance and animal experiment procedure strictly followed ‘Principles of Laboratory Animal Care’ (NIH Publication No. 85–23, revised in 1985) and also local ‘ethical regulations’. (iii) The third group was cultured brain tumor cells (C).
Induction of brain tumor with ENU: N–N–ethyl nitrosourea (ENU) was freshly prepared by dissolving 10 mg/ml in sterile saline and adjusting the pH to 4.5 with crystalline ascorbic acid. ENU was injected intraperitoneally (i.p.) to newborn rats (3–5 days old) with a dose of 80 mg/kg body weight [7-10]. Tumor cell isolation: Four to five consecutive 5 µm sections were cut from the tumor susceptible area of the rat brain and the central section was stained by haematoxyline and eosin. Completely neoplastic areas as appropriate to the histopathological diagnosis and not contaminated by the normal tissue were marked out in the stained slide by the experienced pathologist and the same area were microdissected from the other slide and the scrapings were stored [11]. Then the microdissected tumor mass were dissociated by enzyme digestion {collagenase (0.03%) and DNase (0.01%)} in Hank’s BBS. Finally the cell suspension were centrifuged at 80g for 10 min and thereafter redispersed in cell culture medium containing 10% FBS. A drop of cell suspension was microscopically examined to rule out the possibility, that either because of the nature of the selected tissue or as a consequence of the isolation procedure it is contaminated with the normal brain cells or cellular debris [12-14]. Cell Culture: After isolation of the tumor cells as mentioned above, 1x106 cells/ml of tumor cells were plated on poly-D-lysine-coated 15 cm2 T-flasks in RPMI 1640 media containing 10% heat inactivated FBS, 10U/ml penicillin, 10mg/ml, streptomycin (RPMI 1640 medium, fetal bovine serum, streptomycin and penicillin were purchased fr om Gibco BRL Gaithersburg, MD), at 37°C in a humidified incubator with 4% CO2 for 7 days replacing the medium every 3 days [15]. After 7days the cultured cells were given 2 washes of PBS and viewed under an inverted microscope to check the density of the cells. Maintaining the cell density at 106-107 cells/ml the cells were again fed and placed in the incubator. For 12 weeks the serial culture was maintained replacing the medium at every 3 days. On every 5th day the cultured cells were viewed under microscope to check their growth and density. After 12th week on the day of the experiment, the cells were taken washed twice with PBS, and a culture cell suspension was prepared for the experiment. Antibodies: Rat monoclonal antibody specific for CD95(Fas), CD178 (Fas L), Bcl-2 wer e purchased from BD Biosciences, USA. Rat
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Bhattacharjee et al. monoclonal antibody for Bax was purchased from Santacruz Biotechnology, Santacruz, California. Rat monoclonal antibody specific for Caspase-8 raised against epitope mapping at the N terminus caspase 8 p20 subunit of human origin used for detection of the precursor of caspase -8 were pur chased fr om Santacr uz Biot echnology, Santacruz, California. Rat monoclonal antibody specific for Caspase-3 raised against epitope corresponding to amino acids 1-277 representing the full-length precursor form of caspase 3 of human origin and rat monoclonal antibody specific for Caspase-9 raised against epitope corresponding to amino acids 315-397 mapping within the Cterminus of caspase-9 of human origin used for detection of the precursor of caspase -9 were pur chased fr om Santacr uz Biot echnology, Santacruz, California. The directly labeled antibody employed was mouse FITC-conjugated anti-rat p53 (Pharmingen, BD Biosciences). The secondary antibody employed were FITC-conjugated sheep anti-mouse IgG1 (Pharmingen, BD Biosciences). The primary and the secondary antibodies were diluted in PBS/azide (1% FBS 1%sodium azide in PBS) plus 10 % autologous rat serum [16]. Labeling o f cells and Flow cytometric analysis: Single color analysis was used for all the antibodies. For the surface antigens CD95 and CD178, 1x106 cells/ml were directly incubated with the primary antibodies for 45 mins at 4°C. The cells were then washed and resuspended in PBS after which they were stained with the secondary antibody FITC, in the next step (30 mins. at 4°C in dark). After washing, cells were fixed with 1ml ice-cold 1% paraformaldehyde in PBS (pH 7.2) overnight at 4°C in dark. For intracellular antigens Bcl-2, Bax, p53, caspase-8, caspases-9 and Caspase-3, 4 batches of 1x106 cells/ml were fixed in 0.5 ml of 0.3% ice cold paraformaldehyde in PBS (30 mins at room temperature). Cells were then washed with PBS and permeabilized by being gently resuspended in 1% Triton-X 100 in PBS and incubated at 37°C for 30 mins. The samples were then labeled with the corresponding primary and secondary antibodies and fixed as described above. After overnight incubation, samples were taken out and resuspended in PBS and kept in ice on dark and analyzed within 1 hour. Immunofluorescence was performed in a FACS caliber using Expo 32 software (Beckman Coultier, USA). For each sample, 40,000 events were scored. For surface and intracellular antigen labeling, the Ig-
control sample values were subtracted from all other sample values to remove FITC background fluorescence. RESULTS 1. Differential expression of the cell surface receptor: Fas (CD178): FACS analysis showed a difference in the level of Fas/FasL expression in the 3 groups viz. normal brain cells (NBc), freshly isolated (in vivo) glioma cells (IGc) and cultured glioma cells (CGc). Maximum significant (p
