Background

 

Leukaemia is a progressive disease resulting from the production of abnormal leukocytes (Andreeff, 2014). Research indicates that in the United Kingdom, there are approximately 275,000 people that are diagnosed with cancer, with around 150,000 individuals dying every year (Maddams, 2008), a number that has been decreasing. Leukaemia makes the bone marrow to produce abnormal white blood cells, which fail to function as required. Treatment of leukaemia depends on different factors such as the kind of leukaemia, . The various kinds of Leukaemia include acute lymphoblastic leukaemia (ALL), Chronic myeloid leukaemia (CML), Acute myeloid leukaemia (AML) and Chronic lymphocytic leukaemia (CLL).

 

There are many types of cancer treatments. However, these depend on the disease category and its advancement. Some of the treatment methods include:

 

The hBCATs (human branched-chain aminotransferase proteins) are responsible for catalysing the reversible transamination of the branched-chain amino acids’ α-amino group to the α-ketoglutarate form. This leads to the formation of their particular glutamate and α-keto acids with branched chain (Testa, Caraceni & Fetoni, 1989). There is further metabolism of the branched chain α-keto acids through decarboxylation that is irreversible. The branched chain BCKDH (keto acid dehydrogenase complex) acts as a catalyst of this process. Even though there are several isoforms of hBCAT, the two predominant isoforms are the mitochondrial (hBCATm) and the cytosolic (hBCATc). The hBCATm is mostly expressed in the , whereas the hBCATc is mainly expressed in the peripheral nervous system and the brain (O’Connell, 2013). hBCAT proteins function as “homodimers” with hBCATm having a 41.73 kDa molecular mass and hBCATc 43.40 kDa (Testa, Caraceni and Fetoni, 1989). The brain’s de novo glutamate (>30%) is contributed by the BCAT proteins’ transamination in the brain.  In rat and human brain, the specificity of (Testa, Caraceni & Fetoni, 1989).  In this case, the expression is primarily in the proximal dendrites and the soma. It maintains its function of contributing to the metabolic pools and the storage of glutamate. This is, sequentially, processed to GABA through decarboxylation that takes place in GABAergic neurons (O’Connell, 2013).

 

The Branched chain amino acids (BCAAs) have been identified as key in cancer therapy. The BCAAs, predominantly leucine, convey anabolic properties on the metabolism of proteins as they elevate the synthesis of proteins and also, decrease the rate at which proteins are degraded in latent muscles, hence reducing the signalling of the cancerous cell (Shin et al., 2013). BCAAs play a critical role in the metabolic process as they enhance the functionality of the mitochondria. This facilitates energy production (O’Connell, 2013). Moreover, BCAAs have been linked with a critical role in the signalling pathways, mainly the Mechanistic Target of Rapamycin (mTOR). mTOR is vital in detecting nutrients and also aid in their uptake. Conversely, a number of clinical disorders have been attributed to defective branched-chain amino acid transamination. These include hypervalinaemia, hyperleucine-isoleucinaemia and to a greater degree, carcinogenesis (Sanchez, Gil and Perán, 2015).

 

Hypervalinaemia is a disorder characterized by the serum levels of the BCAA valine being elevated relative to other BCAAs. Distortions experienced during the metabolism of BCAAs results in neurological dysfunctions such as stroke and liver disease. Gene mutation is the major cause of BCAAs disorder. Acute myelogenous leukaemia (AML) is one of the clinical disorders associated with BCAAs (O’Connell, 2013). Most of the population diagnosed with leukaemia have AML. Several diagnostic mechanisms that seek to address AML involve the administering of cytotoxic chemotherapies such as Antimetabolites, Alkylating agents and Anthracyclines (O’Connell, 2013).

 

However, medical concerns are often raised regarding to the effects of chemotherapy and the development of a medical intervention that addresses AML, while reducing the side effects is desirable. Survival and proliferation of leukaemia can be reduced by the use of small molecular substances (such as BCAAs) to inhibit signalling of the cancerous tissues with healthy tissues (Rowley, 1973). mTOR together with protein kinase B (AKT) and phosphoinositide 3-kinase are key in the regulation of AML cells signalling. This can be used to inhibit the oncogenic pathway of leukaemic cells (Karthik et al., 2015). BCAAs are vital in the inhibition of malignant cell growth and facilitate containment of leukaemia cells by limiting their signalling with health cells (O’Connell, 2013).

Aim and objectives

1. Specific aim 1: Investigate the protein expression of hBCAT in leukemic cell lines such as TK6, U261. RPMT, TIM3, Jurkat, k562 and OMLP-4.
   1. Sub aim: Monitor expression after treatment with chemotherapeutics (e.g. Melphalan, Carmustine, Etoposide and Vincristine).
2. Specific aim 2: Investigate the cell surface expression of the hBCAT proteins in leukaemic cell lines
   1. Sub aim: Monitor expression after treatment with chemotherapeutics
3. Specific aim 3: Investigate genetic expression of hBCAT.

 

Main hypothesis:

The hBCAT protein is expressed within leukaemic cell lines and that this expression is sensitive to chemotherapeutic treatment (Melphalan, Carmustine, Etoposide and Vincristine).

 

 

Methodology

Cell culture of TK6, RPMI, MOLP-4, JURKAT, and K562 cells

            The TK6, RPMI, MOLP-4, JURKAT, and K562 cells will be cultured in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin antibiotics (complete medium). Cells will be incubated at 37 ºC in a 5% CO₂ atmosphere.

 

Cell viability (MTS) assay

MTS Cell Proliferation Assay is a colorimetric assay that is used to determine the viability of cells in proliferation or cytotoxic assays. Viability of the TK6, RPMI, MOLP-4, JURKAT, and K562 cells will be measured using the MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (CellTiter 96, Promega, Madison). All cell lines will be seeded in a 96-well plate using complete medium at a density of 5×10⁵ cells/well. Cells will be treated with different concentrations of Melphalan, Carmustine, Etoposide, and Vincristine. The cells will be incubated with the agents for 72 hours. Then, the MTS reagent will be added and cells will be incubated for 2 hours. Using a spectrophotometer, cell viability will be measured using the .

 

Quantification

Total cell proteins will be quantified using Bradford protein assay (Bradford, 1976). The TK6, RPMI, MOLP-4, JURKAT, and K562 cells will be lysed in lysis buffer (1% sodium dodecyl sulphate solution). The protein extract will be denatured in sample buffer (at 95oC) and separated by 12% acrylamide SDS-PAGE

 

Real Time PCR

hBCAT mRNA will be measured in response to Melphalan, Carmustine, Etoposide, and Vincristine or the control, using real time PCR. Specific primers for BCAT will be used in this assay and the results will be done using PCR program. The PCR specificity will be measured using agarose gel for each sample.

 

Flow cytometry

The surface expression of hBCAT proteins in the TK6, RPMI, MOLP-4, JURKAT, and K562 cells will be detected by flow cytometry. An MFC (Multiparameter flow cytometry) will be utilized in assessing hBCAT proteins expression following induction psychoanalysis (n-378) and at weeks 7-8 after treating the cell lines with chemotherapeutics. This will be followed with the treatment of the cells with varying concentrations of chemotherapeutics and monitoring the cell surface expression of hBCAT with flow cytometry.

 

Statistics analysis

Data will be reported as mean ± SD. Comparisons between multiple groups will be performed with one-way analysis of variance (ANOVA) and Tukey post-hoc analysis to compare the results. Results with a p value less than 0.05 will be considered statistically significant and will be calculated using SigmaPlot ® software.

 

Time framework

Key:

Weeks 1-2 (January): Learning cell culture of your various cell lines including both adherent and non-adherent cell lines. In addition to this, will be making buffers for future techniques.

Weeks 2-3 (from January to February): Learning how to perform Western blot on cell lines.

Weeks 4 (February): Performing Western blot on samples to determine the level of hBCAT in these cell lines.

Weeks 5-7 (from February to March): Treating the cells with varying concentrations of chemotherapeutics and monitoring the expression of hBCAT with Western blot.

Weeks 7-8 (March): Learning flow cytometry to investigate cell surface expression of the hBCAT protein.

Weeks 8-10 (March to April): Treating cells with varying concentrations of chemotherapeutics and monitoring the cell surface expression of hBCAT with flow cytometry.

References

Andreeff, M. 2014. Targeted Therapy of Acute Myeloid Leukemia. Berlin: Springer.

Bjorkstrand, B., et al., 1996. Allogeneic Bone Marrow Transplantation versus Autologous Stem Cell Transplantation in Multiple Myeloma: Retrospective case matched study from European Group for Blood and Marrow Transplantation. Blood, 88(12), pp.4711-471

Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Ana1 Biochem, 72, pp.248-253.

Entwistle, G., & Rees, T., 1988. Enzymic capacities of amyloplasts from wheat (Triticum aestivum) endosperm. Biochem J, 255(2), pp.467-472.

Karthik, G., et al., 2015. mTOR inhibitors counteract tamoxifen-induced activation of breast cancer stem cells. Cancer Letters , 367(1), pp.76-87.

O’Connell, T. M., 2013. The Complex Role of Branched Chain Amino Acids in Diabetes and Cancer. Metabolites, 3(4), pp.931-945.

Rowley, J. D., 1973. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature, 243, pp.290-293.

Sanchez, F., Gil, J.  G., and Perán, M., 2015. Modifications of L-Glutamine and L-Leucine Transport in Proliferating Lymphocytes. Journal of Biosciences and Medicines, 3(3), pp.104-109.

Shin, J., et al., 2013. Mechanistic target of rapamycin complex 1 is critical for invariant natural killer T-cell development and effector function. Proceedings of the National Academy of Science, 111(8), pp.E776- E783.

Testa, D., Caraceni, T., and Fetoni, V., 1989. Branched-chain amino acids in the treatment of amyotrophic lateral sclerosis. J Neurol. 236 (8), pp.445-7.