Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.

Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.

Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy

Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.

Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties

In this study, we evaluated the biological properties of human mesenchymal stem cells transfected (hMSC) with a plasmid vector expressing human cytokine interleukin-12 (IL-12). Surface markers were analysed by immunophenotyping using flow cytometry. Differentiation capability was evaluated towards adipogenesis and osteogenesis. We demonstrated that successfully transfected hMSC retained their surface immunophenotypes and differentiation potential into adipocytes and osteocytes. These results indicate that hMSC may be a suitable vehicle for gene transduction.

Dendritic cell distribution in lymphomas

Dendritic cells (DC) are specialized antigen presenting cells (APC) that have important roles in host defenses and in generating anti-tumour immune response. Altered frequency and maturation of DC have been reported in malignant tumours. We studied the distribution and maturation status of DC by immunohistochemistry, on the formalin-fixed, paraffin-embedded lymph node tissues of 32 histologically diagnosed lymphomas and 40 inflammatory conditions that were retrieved from the Department of Pathology, UKM Medical Centre, Kuala Lumpur. Our study showed a significant reduction in the total DC counts in the lymphoma tissues compared to the inflammatory conditions. The mature and immature DC counts were both significantly reduced (p = 0.008 and 0.001 respectively), although a greater reduction was observed in mature DC numbers. We also observed compartmentalization of DC where the immature DC were seen within the tumour tissues and the mature DC were more in peri-tumoural areas. Our findings were similar to other reports, suggesting that reduced numbers of DC appears to be a factor contributing to lack of tumour surveillance in these cases.

Molecular responses during chemotherapy in acute myeloid leukemias in predicting poor-response to standard chemotherapy

Signal transduction pathways are constitutively expressed in leukaemic cells resulting in aberrant survival of the cells. It is postulated that in cells of chemo-sensitive patients, chemotherapy induces apoptotic signals leading to cell death while survival signals are maintained in cells of chemo-resistant patients. There is very little information currently, on the expression of these mediators in patients immediately after chemotherapy initiation. We examined the expression pattern of proinflammatory cytokines, signaling molecules of the PI3K and MAPK pathways molecules and death receptor, DR5 on paired samples at diagnosis and during chemotherapy in acute myeloid leukaemia patients treated with cytosine arabinoside and daunorubicin. The results were correlated with remission status one month after chemotherapy. We found that in chemo-sensitive patients, chemotherapy significantly increased the percentage of cases expressing TNF-alpha (p = 0.025, n = 9) and IL-6 (p = 0.002, n = 11) compared to chemo-resistant cases. We also observed an increased percentage of chemo-sensitive cases expressing DR5 and phosphorylated p38, and Jnk. Thus, expression of TNF-alpha, IL-6, DR5, phospho-p38 and phospho-Jnk may regulate cell death in chemo-sensitive cases. In contrast, a significantly higher percentage of chemo-resistant cases expressed phospho-Bad (p = 0.027, n = 9). IL-beta and IL-18 were also found to be higher in chemo-resistant cases at diagnosis and during chemotherapy. Thus, expression of various cellular molecules in leukaemic blasts during chemotherapy may be useful in predicting treatment outcome. These cellular molecules may also be potential targets for alternative therapy.