Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia.

BACKGROUND: The molecular changes in vivo in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response. METHODS: The gene expression was examined by whole genome 44 k oligo microarrays and 12 k cDNA microarrays in peripheral blood leukocytes collected from seven leukemia patients before treatment, 2-4 h and 18-24 h after start of chemotherapy and validated by real-time quantitative PCR. Statistically significantly upregulated genes were classified using gene ontology (GO) terms. Parallel samples were examined by flow cytometry for apoptosis by annexin V-binding and the expression of selected proteins were confirmed by immunoblotting. RESULTS: Significant differential modulation of 151 genes were found at 4 h after start of induction therapy with cytarabine and anthracycline, including significant overexpression of 31 genes associated with p53 regulation. Within 4 h of chemotherapy the BCL2/BAX and BCL2/PUMA ratio were attenuated in proapoptotic direction. FLT3 mutations indicated that non-responders (5/7 patients, 8 versus 49 months survival) are characterized by a unique gene response profile before and at 4 h. At 18-24 h after chemotherapy, the gene expression of p53 target genes was attenuated, while genes involved in chemoresistance, cytarabine detoxification, chemokine networks and T cell receptor were prominent. No signs of apoptosis were observed in the collected cells, suggesting the treated patients as a physiological source of pre-apoptotic cells. CONCLUSION: Pre-apoptotic gene expression can be monitored within hours after start of chemotherapy in patients with acute myeloid leukemia, and may be useful in future determination of therapy responders. The low number of patients and the heterogeneity of acute myeloid leukemia limited the identification of gene expression predictive of therapy response. Therapy-induced gene expression reflects the complex biological processes involved in clinical cancer cell eradication and should be explored for future enhancement of therapy.

Cyclin B1 is commonly expressed in the cytoplasm of primary human acute myelogenous leukemia cells and serves as a leukemia-associated antigen associated with autoantibody response in a subset of patients.

OBJECTIVES: Aberrant expression of cyclin B1, a cell cycle regulator, is related to prognosis in various human malignancies. Additionally, cytoplasmic expression of cyclin B1 in epithelial malignancies is associated with a specific T-cell response and presumably also a humoral immune response. We therefore investigated (i) whether a similar expression pattern could be detected in native human acute myelogenous leukemia (AML) cells and (ii) whether cyclin B1 specific antibodies could be detected in AML. METHODS: AML cell expression of cyclin B1 was analyzed by flow cytometry and confocal microscopy. Humoral immune response in AML patient sera against cyclin B1 was analyzed by ELISA. RESULTS: AML cell expression of cyclin B1 was detected for all 42 patients; but the percentage of cyclin B1 positive cells showed a wide variation between patients. Confocal microscopy demonstrated that 32/42 (76%) patient samples showed abnormal cytoplasmic expression. Furthermore, the cytoplasmic expression was maintained after 14 d of in vitro culture and differentiation of the AML cells towards a dendritic cell phenotype. Cyclin B1 specific serum antibodies could be detected for seven of 65 patients with untreated AML. CONCLUSIONS: Our studies demonstrate that primary human AML cells show aberrant cytoplasmic expression of cyclin B1 for a majority of patients and a specific humoral immune response was also detected for a subset of patients with untreated leukemia.

Early gene expression of acute myeloid leukemia in response to chemotherapy.

The use of gene expression arrays in the evaluation and classification of tumors is becoming increasingly important in a number of malignancies. This is a powerful technique able to disclose interpatient variance in gene expression. Such variation in gene expression may be the cause of different disease outcome and may reflect disease phenotypes or chemoresistance. Acute myeloid leukemia is a malignant disease of the bone marrow where overall long-term disease-free survival is less than 50%. The need for better disease classification and evaluation is consequently evident. Gene expression profiling in acute myeloid leukemia has, in recent years, proven able to distinguish acute myeloid leukemia subclasses and predict clinical outcome and is, as such, a promising technique for improved disease evaluation. The early detection of gene expression in response to chemotherapy may be a novel way of monitoring disease management. The immediate gene response may be an indication of whether the drug of choice is efficient in leukemic cell eradication and may early indicate the need for other therapeutic measures. Furthermore, these early alterations in gene expression could facilitate identification of new treatment targets, thereby enabling better patient care and follow-up in the future.

Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53.

Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity.

Bcl-2 antisense in the treatment of human malignancies: a delusion in targeted therapy.

Regulation of cell death (apoptosis) is frequently affected in the development of malignant diseases, and all molecular steps from extracellular signalling receptors through intracellular pathways, cell death rheostats and cell death executioners may be involved. Bcl-2 is an anti-apoptotic member of a family of anti- and pro-apoptotic proteins that is upregulated in a variety of cancers and specifically overexpressed through chromosomal translocation in some non-Hodgkin lymphomas. Experimental attenuation of Bcl-2 lowers the threshold for undergoing chemotherapy-induced apoptosis. Therefore, therapeutic targeting of Bcl-2 appears as an attractive approach currently intensely explored using mRNA degradation strategies and small inhibitory molecules. One phosphorothioate oligodeoxynucleotide antisense against Bcl-2 mRNA, oblimersen (Genasense, G3139), has been used in a substantial number of clinical trials. In this review we will discuss the current developments of G3139, and scrutinize its proposed mechanism of action. Several studies indicate that G3139 involves various intracellular mechanisms and modulation of the immune system. To this date G3139 has not been justified in cancer therapy due to modest or absent effects. But, surprisingly, some of its off-target effects may represent useful therapeutic principles. Therefore, antisense uptake improvements and new design of the oligonucleotide may provide us with useful therapeutics, including both the targeted gene and new anticancer mechanisms. This may be another example of how targeted therapy molecules evolve into multimodality drugs when moved from laboratory bench to bedside use, and illustrate our limited ability for target prediction and scant understanding of biological systems when designing therapeutic strategies.

Adaptive contrast enhancement of two-dimensional electrophoretic protein gel images facilitates visualization, orientation and alignment.

2-DE is a powerful technique to discriminate post-translationally modified protein isoforms. However, all steps of 2-DE preparation and gel-staining may introduce unwanted artefacts, including inconsistent variation of background intensity over the entire 2-DE gel image. Background intensity variations limit the accuracy of gel orientation, overlay alignment and spot detection methods. We present a compact and efficient denoising algorithm that adaptively enhances the image contrast and then, through thresholding and median filtering, removes the gray-scale range covering the background. Applicability of the algorithm is demonstrated on immunoblots, isotope-labeled gels, and protein-stained gels. Validation is performed in contexts of (i) automatic gel orientation based on Hough transformation, (ii) overlay alignment based on cross correlation and (iii) spot detection. In gel stains with low background variability, e.g. Sypro Ruby, denoising will lower the spot detection sensitivity. In gel regions with high background levels denoising enhances spot detection. We propose that the denoising algorithm prepares images with high background for further automatic analysis, without requiring manual input on a gel-to-gel basis.

A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia.

PURPOSE: The antioncogene protein product p53 has not been studied previously in cancer patients during in vivo chemotherapy. This study examined the early p53 protein and gene expression during induction chemotherapy in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Leukemic cells were collected from five AML patients during their first 18 hours of induction chemotherapy and examined for p53 protein and gene expression by one- and two-dimensional gel immunoblot and high-density gene expression arrays. RESULTS: Up-regulation of p53 protein expression was detected in AML patients posttreatment in vivo. One- and two-dimensional gel immunoblots showed two main forms of p53, denominated alpha p53 and delta p53, both recognized by various NH2-terminal directed antibodies. As a response to treatment, we detected rapid accumulation of alpha p53, with significantly altered protein expression levels already after 2 hours. The accumulation of alpha p53 was accompanied by increased transcription of putative p53 target genes and subsequent cytopenia in the patients. CONCLUSION: Up-regulation of the p53 protein and target genes seems to be a prominent feature in induction chemotherapy of AML. The rapid shift from a shorter p53 protein form (delta) toward the full-length protein (alpha) underscores the complexity of p53 protein modulation in patients undergoing chemotherapy.

Proteomics of p53 in diagnostics and therapy of acute myeloid leukemia.

The anti-oncogene TP53 is frequently mutated in human cancer, but in hematological malignancies this is a rare feature. In acute myeloid leukemia (AML) more than 90% of the patients comprise wild type TP53 in their cancer cells, but if TP53 is mutated or deleted the disease is often found to be chemoresistant. In this review we define proteomics of the oncogene product p53 as the study of proteins in the p53 regulating signaling networks, as well as the protein study of members of the p53 family itself. Various messenger RNA splice forms as well as a multitude of post-translational modifications give a high number of protein isoforms in the p53 family. Some of the proteomic techniques allow detection of various isoforms, such as two-dimensional gel electrophoresis in combination with tandem mass spectrometry (MS/MS) and this methodology may therefore increasingly be used as a diagnostic tool in human disease. We introduce the p53 protein as an illustration of the complexity of post-translational modifications that may affect one highly connected protein and discuss the possible impact in AML diagnostics if the p53 profile is reflecting cell stress and status of signal transduction systems of the malignancy.

Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables.

BACKGROUND: Two-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance. RESULTS: We validated this technique on a set of simulated 2DE images, which revealed also correct working under the presence of normalization factors. This was followed by an analysis of p53 2DE immunoblots from cancer cells, known to have unique signaling networks. Since p53 is altered through these signaling networks, we expected to find correlations between the cancer type (acute lymphoblastic leukemia and acute myeloid leukemia) and the p53 profiles. A second correlation analysis revealed a more complex relation between the differentiation stage in acute myeloid leukemia and p53 protein isoforms. CONCLUSION: The presented analysis method measures relations between 2DE images and external variables without requiring spot detection, thereby enabling the exploration of biosignatures of complex signaling networks in biological systems.

Protein lysine acetylation in normal and leukaemic haematopoiesis: HDACs as possible therapeutic targets in adult AML.

Several new therapeutic strategies are now considered for acute myelogenous leukaemia (AML), including modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs): a large group of enzymes that alters the acetylation and, thereby, the function of a wide range of nuclear and cytoplasmic proteins. Firstly, HDACs can deacetylate histones as well as transcription factors, and can modulate gene expression through both these mechanisms. Secondly, acetylation is an important post-translational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis (e.g., p53, tubulin, heat-shock protein 90). The only HDAC inhibitors that have been investigated in clinical studies of AML are butyrate derivatives, valproic acid and depsipeptide. In the first studies, the drugs have usually been used as continuous therapy for several weeks or months, and in most studies the drugs were used alone or in combination with all-trans retinoic acid for treatment of patients with relapsed or primary resistant AML. Neurological toxicity and gastrointestinal side effects seem to be common for all three drugs. Complete haematological remission lasting for several months has been reported for a few patients (< 5% of included patients), whereas increased peripheral blood platelet counts seem more common and have been described both for patients with AML and myelodysplastic syndromes. Taken together, these studies suggest that HDAC inhibition can mediate antileukaemic effects in AML, but for most patients the clinical benefit seems limited and further studies of combination therapy are required.

Proteomics in acute myelogenous leukaemia (AML): methodological strategies and identification of protein targets for novel antileukaemic therapy.

Enduring efforts into determination of the molecular biological status of acute myelogenous leukaemia (AML), a stem cell disease characterised by distinct blastic differentiation blocks and their extensive growth, continue to provide us with prognostically important information for more than half of all patients. In subsets of AML, molecular diagnostics rigorously guide the clinician toward the choice of optimal therapy. The in-depth characterization of leukemogenesis associated genetic alterations, such as the combined presence of activating mutations of tyrosine kinases together with altered transcription factors, and the documented impact of these mutations upon prognosis of AML, suggests AML as a primary candidate for pioneering proof-of-principle studies with new high throughput protein analysis techniques. This review aims to introduce the reader to proteomic methodology, e.g. two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, SELDI and protein arrays. Examples of its use, including single cell phosphoprotein profiling in risk stratification, the probing of cellular effects of conventional chemotherapeutics and novel target determination are presented. Based on original proteomic analysis of AML, molecular characteristics of AML, in addition to knowledge of conventional therapeutics and novel drugs, we attempt to forecast the influence of proteomics in therapy development for AML.