Semi-quantitative RT-PCR analysis to study overexpression of MCSF gene in U87-MCSF cells. U87-MCSF cells were treated with 5-FU for 72 hours and stained with AO/EB double staining solution. U87MG and U87-MCSF cells were treated with 5-FU for 72 h and the expression of Caspase-3, Bax and Bcl-xL was examined.
Expression of p21, a cyclin-dependent kinase inhibitor, was increased in treated U87MG and U87-MCSF cell samples. The results showed a decrease in the expression of cyclin E in the treated samples of U87MG and U87-MCSF cells. However, treatment with 50 µM 5-FU produced an elongated morphology in treated U87MG and U87-MCSF cell samples.
No change in GFAP expression was observed between untreated and treated U87MG and U87-MCSF cell samples. Upregulation of the expression of mesenchymal markers, N-cadherin and vimentin, was observed in untreated and treated U87-MCSF cells.
Immune system and Cancer
Immune system and cancer, section 1, are highly resistant to chemotherapeutic drugs and have high expression of MDR-associated proteins. They are quiescent, with a slow proliferation rate (Moore and Lyle, 2011) and can exhibit both symmetric and asymmetric cell division (Clarke et al., 2006). These overlapping properties between normal stem cells and cancer stem cells make it a daunting task to develop a targeted therapeutic regimen for cancer stem cells.
Any treatment method designed to target cancer stem cells also carries the risk of destroying normal stem cells. Our current understanding of cancer stem cell biology is limited and therefore further insights into the differences in the physiological and immunological aspects of cancer stem cells are needed to identify drug targets unique to cancer stem cells. Furthermore, the tumor microenvironment is reported to have a heavy infiltration of cells of the innate immune system, especially macrophages, and these tumor-associated macrophages (TAM) are known to promote tumor growth and metastasis ( Condeelis and Pollard, 2006 ; Sica et al., 2008).
Macrophage Colony Stimulating Factor
Objectives
Salient features of the present work
Salient features of the present work Chapter 1 underwent an epithelial-mesenchymal transition, which ultimately culminated in the increase of the cancer stem cell population and the increase in resistance of U87-MCSF cells to 5-FU treatment.
R EVIEW OF L ITERATURE 11-30
- Colony Stimulating Factors
- Macrophage Colony Stimulating Factor (MCSF or
- MCSF receptor and signaling cascades
- Physiological functions of MCSF
- MCSF and cancer
- Cancer stem cells
- Implications of CSCs in drug resistance
All isoforms of MCSF are biologically active and can stimulate cell proliferation on target cells (Stein et al., 1990; Wang et al., 1993). In addition to PI3K-mediated signaling, phosphorylated Y721 also recruits PLCγ2 (Bourette et al., 1997) which cleaves phosphatidylinositol 4, 5-bisphosphate (PIP2) into two products, namely diacylglycerol (DAG) and (3IP, 41). . Furthermore, MCSF has also been reported to be a crucial factor required for normal fertility (Pollard et al., 1991).
Overexpression of MCSF and its receptor CSF-1R in tumors has been associated with poor prognosis (Chambers et al., 1997). MCSF and cancer 2. transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) (Sica et al., 2006; Richards et al., 2013). Tumor-associated macrophages (TAMs) often accumulate in low-oxygen and avascular regions within the tumor (Murdoch et al., 2004; Coffelt et al., 2009).
MCSF and cancer Section 2 and increased invasive properties are observed in ovarian cancer cells (Chambers et al., 1995). It is also possible that tumor cells undergo the process of de-differentiation by cellular fusion with stem cells (Bjerkvig et al., 2005).
M ATERIALS A ND M ETHODS 31-54
Plastic and glass wares
PCR tubes were from Greiner, micro tips, centrifuge tubes, petri dishes for bacterial cultures and other common plastic products were procured from Tarsons Products Pvt. Plastics used for mammalian cell culture work were purchased from Greiner bio-one, Corning and BD Falcon, BD Biosciences, India.
List of Bacterial strains
Mammalian cell lines
Media, solutions and buffers
LB agar media was prepared using 1.5% Agar in Luria Bertani broth and sterilized by autoclaving at 121 °C, 15 psi for 20 minutes. LB agar plates were made by pouring 20 ml of LB agar media with desired antibiotics into Petri dishes. Dulbecco's Modified Eagles Media (DMEM) for mammalian cell culture DMEM media was prepared by mixing 13.47 g of high glucose DMEM powder, 3.75 g of Sodium Bicarbonate and Penicillin (50U/ml)- Streptomycin (50mg/ml) solution in 1 to add. liter of double distilled water.
For routine mammalian cell culture, DMEM media was supplemented with 10% Fetal Bovine Serum (FBS). The phosphate buffer contained 140 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate.
Methods 41-53
- Isolation of plasmid DNA from bacterial
- Quantification of DNA and RNA
- Agarose gel electrophoresis
- Restriction enzyme digestion of DNA…
- Elution of DNA from agarose gel…
- Ligation of DNA
- Preparation of DH5α competent cells
- Transformation of DNA into DH5α
- Polymerase chain reaction
The expression of transgene MCSF in U87-MCSF cells was confirmed by semi-quantitative RT-PCR analysis (Figure 4.5). The expression of anti-apoptotic gene, Bcl-xL, was also increased in treated U87MG and treated U87-MCSF cells. The results showed that the rate of proliferation of untreated U87MG and U87-MCSF cells remained unchanged.
However, treatment with 50 μM 5-FU produced an elongated morphology in treated U87MG and U87-MCSF cell samples. The results showed the presence of elongated cells with intact nuclei in treated U87MG and U87-MCSF cell samples. The morphology of untreated U87MG and U87-MCSF cells was closely examined using an inverted microscope.
Furthermore, expression of the receptor for MCSF, CSF-1R, was down-regulated in U87-MCSF cells. No increase in CD44 expression was noted in treated samples of either U87MG or U87-MCSF cells. This indicates the presence of slowly proliferating and quiescent cells in treated U87MG and treated U87-MCSF cells.
Treatment with 5-FU resulted in delay of cell proliferation in both U87MG and U87-MCSF cells.
