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Siddhartha Sankar Ghosh, Department of Biotechnology, Indian Institute of Technology Guwahati, India for the award of the degree of Doctor of Philosophy. This confirms that the dissertation titled "Molecular Cloning, Expression, Purification and Functional Implications of Recombinant Cytokines" submitted by NIDHI CHAUBEY to the Indian Institute of Technology Guwahati for the award of the degree of Doctor of Philosophy in the Department of Biotechnology is an authentic record of the research work done by done. I am very grateful to my lab members Kohila, Chokalingam, Subhamoy, Archita, Sharmila, Asif, Neha, Amaresh, Upashi, Bandhan, Amit, Shilpa and Deepanjali who have provided me constant support, inspiration and a pleasant working environment.

I dedicate this work to my family members, my brother Nitish, sister Nishi, husband Ajay, mother Smt. My last words of acknowledgment to the Almighty God who gave me the strength to settle in such a way so that I could complete my work properly.

Abstract

Introduction

Recombinant proteins

Combination therapy

Combination therapy is preferred over single therapeutic module to reduce tumor burden (Miles et al, 2002). A recent report showed that combination therapy not only reduced cancer recurrence (Reference No. 77, Website 7) but also significantly improved the survival rate of patients with metastatic melanoma treated with GMCSF (Reference No. 78, Website 8).

Diagram showing the effect of combination therapy

Classification of cytokines
GMCSF (Granulocyte macrophage colony stimulating factor)
IFNγ (Interferon gamma)
PLGA (Poly lactide co glycolide)
Silver Nanoparticles
Tumour progression phases in host
Chemotherapeutic drugs block cell cycle

A very recent study by Falentin-Daudré et al (2012) showed formation of Ag NPs containing antimicrobial coating formed by layer-for-. In another study, Sahoo et al (2011) developed a nanocomposite consisting of p-hydroxyacetanilide (paracetamol) dimer and Ag NPs by reaction of AgNO3 and paracetamol. Studies are being pursued to explore the possible mechanisms of cytotoxicity and possible genotoxicity of Ag NPs in mammalian cells (AshaRani et al.

Similarly, Carlson et al (2008) also demonstrated Ag NPs-induced oxidative stress in alveolar macrophages and found toxicity of Ag NPs. Furthermore, Franco-Molina et al (2010) reported antitumor activity of Ag NPs on MCF-7 human breast cancer cells.

Fig 1.1. Mode of action of autologous tumour vaccine secreting GMCSF cytokine High peritumoral level of GMCSF produced as transgenes, execute a much better anti-tumour response by attracting T lymphocytes, natural killer cells, neutrophils and

Salient features

Enhanced generation of reactive oxygen species in the antimicrobial activity of a three-component iodinated chitosan-silver nanoparticle composite. Poly(lactide-co-glycolide) nanoparticles as a carrier system for delivery of the cysteine protease inhibitor cystatin to tumor cells. Bacterial expression systems for recombinant protein production: E. G-CSF-loaded biodegradable PLGA nanoparticles prepared by a single oil-in-water emulsion method.

Plasmid DNA linearization in the antibacterial effect of a new fluorescent Ag nanoparticle-paracetamol dimer composite. Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF.

Introduction

The intracellular localization of the GST-tagged protein within the bacterial host is unknown, but it may sort the synthesized protein into the periplasmic space, and thus, aid in the delivery of proteins from inclusion bodies under mild conditions (Speers et al, 2007). The GMCSF gene was cloned into the pGEX4T2 bacterial expression vector to generate GST-fused GMCSF. The three-dimensional structure of GST-GMCSF and its GMCSF receptor-binding domain were predicted by in silico modeling and protein-protein binding studies.

Furthermore, the proliferative effect of GST-GMCSF protein on different cell types was found to be remarkably dose-dependent. To our knowledge, this is the first report to demonstrate the effect of GST-tagged GMCSF cytokine in cell proliferation.

An overview of cloning and functional details of human GMCSF

Materials and Methods .1 Cell Culture

Isolation of RNA from mammalian cell lines
Reverse transcription for cDNA synthesis
Quantification of DNA and RNA
Polymerase Chain Reaction
Agarose gel electrophoresis
Gel Elution
Plasmid DNA Isolation (Mini-Prep Method)
Preparation of the Competent Cells
Transformation in bacterial cells
Colony PCR for screening recombinant clones
Digestion by Restriction Enzymes
Ligation reaction
Cloning of PCR amplified DNA fragments in T/A vector system
Bacterial cell culture
Sequencing of DNA fragments cloned in pGEMT Easy Vector
Subcloning of GMCSF in pGEX4T2 Expression vector
Expression of GMCSF in E.coli BL21 DE3 strain
Purification

Solubilisation of inclusion bodies
Refolding and purification

Homology Modelling of GST-GMCSF protein
Western Blot Analysis
MALDI TOF TOF analysis
Circular dichroism analysis
Cell proliferation Assay
Results and Discussion .1 Cloning of GMCSF cDNA
Conclusion
References
Introduction
Materials and Methods 3.2.1 Cell Culture
Results and Discussions .1 Cloning of human IFNγ
Conclusion
References
Introduction
Outline of Work
Materials and Methods .1 Cell Culture
Results and Discussions

The interactions were compared with those of the crystallized structure of GMCSF (3CXE: PDB ID). PDBsom analysis of the secondary structure of the GST-GMCSF protein model showed 48 % α helix and no β sheets. The presence of GS IFNγ protein was confirmed by 12% SDS-PAGE analysis of the cell lysate.

The anti-cell proliferative activity of the recombinant IFNγ was further tested on MCF-7 and HeLa cells using the MTT test. Thus, we were able to deposit Ag NPs on the surface of the PLGA NPs. Interestingly enough; the Ag NPs were clearly visible to be embedded in the PLGA NPs in the case of the composite NPs.

UV- is investigation of the binding of GS IFNγ protein with (A) PLGA only (B) composite NPs (Ag PLGA NPs). FTIR spectra of (A) composite NPs and (B) recombinant GS IFNγ immobilized GS IFNγ Ag PLGA composite NPs. The finding was substantiated by FACS analysis of the cells treated with FITC-loaded GS IFNγ PLGA NPs.

Finally, an MTT assay was performed to assess cell viability in the presence of protein-immobilized composite NPs (Ag PLGA NPs). Therefore, this combination therapy module could enhance the antiproliferative effect of recombinant IFNγ in the presence of a small amount of Ag NPs. Recombinant IFNγ was immobilized on assembled PLGA NPs together with Ag NPs, where PLGA NPs provided protein stability and Ag NPs enhanced the anticancer activity of recombinant IFNγ.

Approximately 50% of the cells were compromised in both cells with a doxorubicin response at 10 µM. Cell cycle analysis of MCF-7 cells treated with recombinant IFNγ showed a marked cell cycle block in the G1 phase of treated cells (Figure 4.3).

Fig 2.2. Subcloning of GMCSF in pGEX4T2 bacterial expression vector. Lane 1:

Cells in coculture experiments

Conclusion

In this chapter, an easy and fast flow cytometry based method was established to find the effect of recombinant cytokines in mixed cell population. Addition of recombinant IFNγ produced significant reactive oxygen species that could damage the MCF-7 cancer cells, but had little effect on the macrophages. In co-culture experiment, addition of recombinant GMCSF protected the damage of macrophages either in the presence of IFNγ or in combination of both.

Moreover, doxorubicin-based co-culture results revealed that macrophages were rescued from the negative effect of drugs by the administration of proliferative GMCSF cytokine. Consequently, this FACS-based approach can be easily employed to test the effect of cytokines or drugs in combination on the cell growth using simple coculture-based experiments.

Introduction

To achieve the appropriate sensitivity of this cell line to chemotherapy, overexpression of this antitumor cytokine IFNγ in U87MG neuroblastoma cells was performed.

Strategy of work in cytokine over expressing cancer cells

Materials and Methods

Cloning of gene into pCI neo mammalian expression vector
Generation of stable cell lines
Extraction of proteins from mammalian cells
Estimation of Protein
Westeren Blot
Cell Viability Assay
Cell Cycle Analysis
Carboxyfluorescein succinimidyl ester (CFSE) assay
Semiquantitative PCR analysis for cyclins study

Results and Discussions

Cloning of GMCS IFNγ in pCI neo mammalian expression vector
Semiquantitative RT-PCR to check overexpression of GMCSF and IFNγ
Western Blots
Cell Viability Assay
Cell cycle analysis
CFSE based doubling time calculation
Analysis of Cyclins

Conclusion
References

After electroporation, the cells were resuspended in 10% FBS in DMEM with antibiotics and seeded in a 6 well plate. For the transfection of IFNγ into pCI neo mammalian expression vector (Promega), first U87MG (1×106) cells were seeded in the plate. Media was discarded from cell culture plate and the cells were washed with PBS to remove the remaining media.

Approximately 500 µl of RIPA buffer was added to a 35 mm plate and the cells were simply removed by repeated pipetting. The overexpression of GMCSF in GMCSF-transfected MCF-7 stable cells and that of IFNγ in IFNγ-transfected U87MG stable cells were monitored by semi-quantitative RT-PCR. The MCF-7 and U87MG cells transfected with pCI neo vector backbone were taken as controls to examine the effect of the desired gene.

A significant difference in cell viability was observed in stable MCF-7 cells transfected with GMCSF compared to control cells. The viability of stable GMCSF-transfected MCF-7 cells was reduced to 25% relative to their non-transfected control cells, indicating that GMCSF-transfected stable cells were more sensitive to chemotherapeutic drugs such as doxorubicin, cisplatin and 5FU (Fig. 5.8A, 5.8B). , 5.8C). The block was more pronounced in stable GMCSF-transfected MCF-7 cells compared with control MCF-7 cells.

FACS-based cell cycle analysis of stable GMCSF-transfected MCF-7 cells compared to control MCF-7 cells (A) Untreated cells (B) 5 FU-treated cells (C) Cisplatin-treated cells and (D) cells treated with Doxorubicin. Analysis of CFSE-based proliferation by doubling time counting through flow cytometry in (A) MCF-7 cells (B) stable MCF-7 cells transfected with GMCSF. In this chapter, the effect of homogenously overexpressed cytokine GMCSF on cancer cells was studied.

Fig 5.5. restriction digestion profile of the recombinant clones with Xho I, Not I (A) Lane 1: 435 bp release of GMCSF from pCI neo GMCSF construct, Lane 2:

Conclusions and Future Prospects

Experimental results showed complementary effects of the composite nanoparticles, where the recombinant IFNγ had blocked the cell cycle and enabled cell surface receptor targeting; on the other hand, the Ag NPs had induced apoptosis. Thus, the combination of the duos led to efficient induction of apoptosis in cancer cells. The combined function of the recombinant cytokines was investigated for proliferation and activation of macrophage cells and the subsequent generation of ROS, which in turn kills the cancer cells.

GMCSF-treated MCF-7 cells were mixed with macrophages and treated with recombinant IFNγ and GMCSF simultaneously. GMCSF allowed macrophages to remain proliferative, while recombinant IFNγ generated ROS to induce cancer cell death. FACS-based analysis showed efficient death of MCF-7 cells in the mixed population, indicating the potential role of these two pairs of recombinant cytokines in controlling cancer cell growth.

To mimic the state of chemotherapy in a solid tumor, cells were treated with doxorubicin under co-culture conditions, which showed significant cancer cell death. This work concluded that such an approach can be explored to treat a solid tumor with chemotherapeutic drugs together with an adjuvant cytokine to enhance the phagocytic effect of macrophages and effectively suppress tumor growth. In Chapter 5, the generation of cell lines overexpressing GMCSF and IFNγ was reported.

The chapter reported the effects of chemotherapy drugs, doxorubicin, 5FU and cisplatin on these two cytokine overexpressing cells. This observation was explained by cell cycle analysis and semi-quantitative measurement of cyclin expression of the drug-treated cells. Delineation of molecular pathways to investigate the use of recombinant GMCSF and IFNγ along with chemotherapeutic agents.

Appendix

Cell lines: All cell lines were procured from National Center for Cell Science, Pune, India. GMCSF Inclusion body Solution I Solution I 10 mM phosphate buffered saline (PBS), 1 mM PMSF, 1 mM EDTA, 1% Triton X-100 GMCSF Inclusion body solution II 50 mM TrisCl, 5 mM EDTA and 2% Triton.

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