Transformation Potency Research Articles

Flavored E-cigarette product aerosols induce transformation of human bronchial epithelial cells

ObjectivesE-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation. Materials and methodsAn aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes. ResultsJamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers. ConclusionsThese studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.

Cell-to-cell natural transformation in Bacillus subtilis facilitates large scale of genomic exchanges and the transfer of long continuous DNA regions.

Natural transformation is one of the major mechanisms of horizontal gene transfer. Although it is usually studied using purified DNA in the laboratory, recent studies showed that many naturally competent bacteria acquired exogenous DNA from neighboring donor cells. Our previous work indicates that cell-to-cell natural transformation (CTCNT) using two different Bacillus subtilis strains is a highly efficient process; however, the mechanism is unclear. In this study, we further characterized CTCNT and mapped the transferred DNA in the recombinants using whole genome sequencing. We found that a recombinant strain generated by CTCNT received up to 66 transferred DNA segments; the average length of acquired continuous DNA stretches was approximately 27 kbwith a maximum length of 347 kb. Moreover, up to 1.54 Mb genomic DNA (37% of the chromosome) was transferred from the donors into one recipient cell. These results suggest that B. subtilis CTCNT facilitates horizontal gene transfer by increasing the transfer of DNA segments and fostering the exchange of large continuous genomic regions. This indicates that the potency of bacterial natural transformation is underestimated using traditional approaches and reveals that DNA donor cells may play an important role in the transformation process in natural environments.

Identity in the disrupted time of COVID-19: Performativity, crisis, mobility and ethics.

The COVID-19 pandemic has resulted in a global cultural crisis, experienced through various losses of everydayness, including particularly restrictions on mobility and the sudden emergence of new fears and anxieties over infection. This paper theorises some of the ways in which that crisis can be understood in cultural and discursive terms, as a rupture in normativity, a disturbance in social relationality and as a state of exception. Drawing on Judith Butler's theories of performativity, the paper investigates how such a cultural rupture can be understood to affect performative subjectivity, identity and selfhood, whereby a breach in normative everydayness prompts the re-constitution of subjectivity itself. The paper explores how the reconfiguration of identity is experienced as corporeal and as a site of anxiety and lost dignity. The final section of the paper draws some initial conclusions about the potency of cultural and identity transformation for new ethics of non-violence, arguing that the obligation to resist norms of mobility and contact is an ethical obligation of necessary cohabitation.

TRA LEGGE E DESIDERIO. L’ANTI-ADAMO DI DELEUZE

Deleuze’s notion of law is often developed within an analysis of Spinoza’s exposition of the Original sin. Adam interprets the God’s prohibition (concerning a negative effect of a bodily link) as a moral law determining his desiring-action. Following the binary logic of differentiation, he appeals to an erroneous dialectical potency of transformation of content into expression. It is an act of a flow marking an over-coding by resonance organizations: a transcendent signifier distributing effects of meaning and introducing exclusions. However, the law’s purpose (what Deleuze calls institution ) is not to assign guilts, but simply to enable micropower relations. It implies a constellation of juxtaposed subsystems. There is thus no opposition between central and outer organizations. Political system is a global whole, a collective mechanism unified and unifying. It brings to light all kinds of partial processes, but not without gaps and displacements.

Erratum: EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

Liposarcoma in the axilla developed from a longstanding lipoma.

Liposarcoma in the axilla developed from a longstanding lipoma.

Metabolic Engineering of Aliphatic Glucosinolates in Hairy Root Cultures of Arabidopsis thaliana

Two cytochrome P450 proteins, CYP79F1 and CYP79F2, are involved in the biosynthesis of aliphatic glucosinolates (GSs) in brassicaceous species. In order to increase aliphatic GS levels in Arabidopsis thaliana hairy root cultures (HRCs) with the aim of biomanufacturing GSs, CYP79F1 and CYP79F2 from either Arabidopsis thaliana or Brassica rapa were first constitutively expressed in stably transformed Arabidopsis plants, and then HRC was induced by additional transformation with Agrobacterium rhizogenes. Gene expression levels and GS profiles were analyzed in HRC in order to validate the impact on aliphatic levels. To estimate the potency of gene transformation on GS content in HRC, GS levels were compared to levels in roots and leaves of transformed plants. According to the data obtained, short-chain aliphatic GSs in plants were raised in three generations after transformation, indicating that expression of transgenes was stable. After HRC induction, transcript levels of CYP79F1 and CYP79F2 rose and short-chain aliphatic GS levels increased by as much as 7-fold compared to the respective HRC wild-type control. However, when compared to levels present in leaves and roots of plants before HRC induction, aliphatic GS levels in HRC remained very low. In addition, expression of CYP79F1 and CYP79F2 also enhanced the content of indole GSs, mainly indol-3-ylmethyl GS and 4-hydroxy-indol-3-ylmethyl GS in HRC. The results presented provide basic information about GS biosynthesis in HRC of Arabidopsis thaliana compared to intact plants, which will help scientists to further optimize GS composition in HRC in the future.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

The signaling and transformation potency of the overexpressed HER2 protein is dependent on the normally-expressed EGFR

Human epidermal growth factor receptor 2 (HER2) belongs to the EGFR family of receptor tyrosine kinases that comprises four members. As opposed to the other family members, HER2 does not require ligand binding for activation. Hence, HER2 molecules can undergo spontaneous dimerization, autophosphorylation and activation of downstream signaling pathways especially under conditions of overexpression, a commonly encountered phenomenon in breast cancer. In this study, we sought to investigate the mechanism by which HER2 musters signaling and transformation potency. We show that HER2 overexpression per se induces a significant increase in basal mitogenic and cell survival signaling, which was augmented by EGF stimulation. Inhibition of the normally expressed EGFR significantly suppressed the ability of overexpressed HER2 to induce enhanced signaling and cell transformation, suggesting that HER2 requires the EGFR and potentially other members to maximize its signaling and transformation potency. The novel observation revealed by prolonged EGF stimulation studies was the biphasic signaling pattern in the presence of HER2 overexpression that suggested the induction of a short-circuited mechanism, permitting sustained signaling. Our results further show that the short-circuited signaling was due to the re-shuttling of internalized receptor molecules to the Rab11-positive recycling endosomes, while suppressing channeling to the LAMP1-positive lysosome-targeting endosomes. Therefore, HER2's oncogenicity is dependent, not only on its constitutively active nature, but also on its ability to muster collaborative signaling from family members through modulation of ligand-induced receptor regulation.

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

BackgroundThe ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells.ResultsTo map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112) and another to the DNA binding domain (DBD), (NES2: 275LWEFIRDILI284). Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions.ConclusionsThese data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.

Whose Entropy: A Maximal Entropy Analysis of Phosphorylation Signaling

High throughput experiments, characteristic of studies in systems biology, produce large output data sets often at different time points or under a variety of related conditions or for different patients. In several recent papers the data is modeled by using a distribution of maximal information-theoretic entropy. We pose the question: ‘whose entropy’ meaning how do we select the variables whose distribution should be compared to that of maximal entropy. The point is that different choices can lead to different answers. Due to the technological advances that allow for the system-wide measurement of hundreds to thousands of events from biological samples, addressing this question is now part of the analysis of systems biology datasets. The analysis of the extent of phosphorylation in reference to the transformation potency of Bcr-Abl fusion oncogene mutants is used as a biological example. The approach taken seeks to use entropy not simply as a statistical measure of dispersion but as a physical, thermodynamic, state function. This highlights the dilemma of what are the variables that describe the state of the signaling network. Is what matters Boolean, spin-like, variables that specify whether a particular phosphorylation site is or is not actually phosphorylated. Or does the actual extent of phosphorylation matter. Last but not least is the possibility that in a signaling network some few specific phosphorylation sites are the key to the signal transduction even though these sites are not at any time abundantly phosphorylated in an absolute sense.

Gene expression of oncogenes, antimicrobial peptides, and cytokines in the development of oral leukoplakia

The aim of this study was to investigate the expression pattern of oncogenes, antimicrobial peptides, and genes involved in inflammation in leukoplakia of the oral cavity compared with healthy gingiva. Biopsies of healthy gingiva (n=20) and leukoplakia (n=20), were obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of alpha-defensin (DEFA) 1/3, DEFA-4, S100-A7, deleted-in-oral-cancer (Doc) 1, interleukin (IL) 1beta, IL-6, IL-8, IL-10, tumor necrosis factor (TNF) alpha, cyclooxygenase (Cox) 2, epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGF) beta1, TGF-alpha, collagen-IA1 (Col-1), and tenascin-c were analyzed by real-time reverse-transcription polymerase chain reaction. The proteins encoded by the different genes were visualized by immunostaining. Compared with healthy gingiva (set as 1), there was an increased gene expression of DEFA-4 (179.2-fold), S100-A7 (25.4-fold), EGF (24.8-fold), TGF-beta1 (25.2-fold), and tenascin-c (34.3-fold) in oral leukoplakia. The expression of IL-1beta and Doc-1 was decreased (0.01-fold and 0.2-fold, respectively). The combination of an increased expression of the antimicrobial peptide DEFA-4, the oncogene S100-A7, EGF, and tenascin-c, and a decreased Doc-1 expression in oral leukoplakia might characterize its potency of malignant transformation. Chronic inflammation seems not to be involved in the development of this lesion.

Maximal entropy inference of oncogenicity from phosphorylation signaling

Point mutations in the phosphorylation domain of the Bcr-Abl fusion oncogene give rise to drug resistance in chronic myelogenous leukemia patients. These mutations alter kinase-mediated signaling function and phenotypic outcome. An information theoretic analysis of the correlation of phosphoproteomic profiling and transformation potency of the oncogene in different mutants is presented. The theory seeks to predict the leukemic transformation potency from the observed signaling by constructing a distribution of maximal entropy of site-specific phosphorylation events. The theory is developed with special reference to systems biology where high throughput measurements are typical. We seek sets of phosphorylation events most contributory to predicting the phenotype by determining the constraints on the signaling system. The relevance of a constraint is measured by how much it reduces the value of the entropy from its global maximum, where all events are equally likely. Application to experimental phospho-proteomics data for kinase inhibitor-resistant mutants shows that there is one dominant constraint and that other constraints are not relevant to a similar extent. This single constraint accounts for much of the correlation of phosphorylation events with the oncogenic potency and thereby usefully predicts the trends in the phenotypic output. An additional constraint possibly accounts for biological fine structure.

Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland

BackgroundAmong the salivary gland carcinomas, carcinoma in pleomorphic adenoma has been regarded as a representative carcinoma type which arises secondarily in the background of a pre-existent benign pleomorphic adenoma. It is still poorly understood how and which benign pleomorphic adenoma cells transform into its malignant form, carcinoma ex pleomorphic adenoma.MethodsWe have established five cell systems from a benign pleomorphic adenoma of the parotid gland of a 61-year-old woman. They were characterized by immunofluorescence, classical cytogenetics, p53 gene mutational analysis, fluorescence in-situ hybridization, and histopathological and immunohistochemical examinations of their xenografts, to demonstrate their potency of secondary transformation.ResultsWe established and characterized five cell systems (designated as SM-AP1 to SM-AP5) from a benign pleomorphic adenoma of the parotid gland. SM-AP1 to SM-AP3 showed polygonal cell shapes while SM-AP4 and SM-AP5 were spindle-shaped. SM-AP1-3 cells were immunopositive for keratin only, indicating their duct-epithelial or squamous cell differentiation, while SM-AP4/5 cells were positive for both keratin and S-100 protein, indicating their myoepithelial cell differentiation. Chromosome analyses showed numeral abnormalities such as 5n ploidies and various kinds of structural abnormalities, such as deletions, translocations, derivatives and isodicentric chromosomes. Among them, der(9)t(9;13)(p13.3;q12.3) was shared by all five of the cell systems. In addition, they all had a common deletion of the last base G of codon 249 (AGG to AG_) of the p53 gene, which resulted in generation of its nonsense gene product. Transplanted cells in nude mice formed subcutaneous tumors, which had histological features of squamous cell carcinoma with apparent keratinizing tendencies. In addition, they had ductal arrangements or plasmacytoid appearances of tumor cells and myxoid or hyaline stromata, indicating some characteristics of pleomorphic adenoma.ConclusionThis study demonstrates in vitro that certain cell types from pleomorphic adenoma are able to clone and survive over a long term and develop subcutaneous tumors in nude mice. The histological features of squamous cell carcinoma from the transplanted cell systems in nude mice might suggest a secondary onset of malignancy from a pre-existing benign adenoma.

Fire and ritual: bark hearths in South-American Tupiguarani mortuary rites

High proportions of bark pieces (up to 85% of the charcoal content) were found in several hearths from Morro Grande archaeological site (Southeastern Brazil). This site, dated between 3220–2840 and 1820–1390 yrs cal BP, is associated to the Tupiguarani Tradition, attributed to supposed ancestors of Tupinambá native populations, who occupied the major part of the Brazilian coast in the XVIth century. Bark hearths, archaeologically associated with the mortuary ritual, were found encircling a funerary urn and associated with ceramic fragments painted with elaborated patterns in black, white, and red. Other hearths, spatially isolated from the funerary area, were associated to fragments of utilitarian non-painted ceramics and therefore attributed to domestic contexts. These ones presented few or no bark fragments. It is clear that bark was intentionally selected as fuel for the funerary hearths. Although bark is related, in historic accounts, as a specialized firewood for ceramics firing, its presence in ritual context has not been previously recorded. In this paper, the anthracological record is discussed in the light of ethnographic and historic accounts. A possible explanation for the presence of bark hearths in funerary context is proposed, suggesting it might be a symbolic parallel with the quotidian: the potency and power of transformation of bark as a fuel would be regarded in a spiritual level, achieving the transformation of the body soul in the revered soul – an Ancestor.

MMPs, TIMP‐2, and TGF‐β1 in the cancerization of oral lichen planus

Matrix metalloproteinases (MMPs) and transforming growth factor-beta1 (TGF-beta1) are important in many physiological and pathological processes. Immunohistochemistry for MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP, MMP-14), tissue inhibitor of matrix metalloproteinase (TIMP)-2, and TGF-beta were performed on normal mucosa, nonatrophic oral lichen planus, atrophic oral lichen planus, and oral squamous cell carcinomas (OSCC) resulting from lichen planus. Expression of MMPs progressively increased from normal mucosa to nonatrophic oral lichen planus, atrophic oral lichen planus, and OSCCs. Immunoscores of MMPs in atrophic oral lichen planus was significantly greater than nonatrophic oral lichen planus. Moreover, immunoscore of MMP-9 of OSCCs was significantly greater than both atrophic and nonatrophic lichen planus. Furthermore, expression of TIMP-2 and TGF-beta1 paralleled increases seen with MMPs. Imbalance between MMPs and TIMPs may be involved in cancerization of oral lichen planus. MMP-2, MT1-MMP, and especially MMP-9 may be useful markers for judging potency of malignant transformation from oral lichen planus.

Long‐term experience with imatinib therapy in chronic phase chronic myelogenous leukemia—Remarkable activity with room for improvement

Long‐term experience with imatinib therapy in chronic phase chronic myelogenous leukemia—Remarkable activity with room for improvement

RET oncogene amplification in thyroid cancer: Correlations with radiation-associated, high-grade malignancy, and genomic instability

Abstract. RET oncogene rearrangement is a well-known molecular alteration observed in papillary thyroid cancer (PTC). RET rearrangement is the commonest oncogenic alterations in Chernobyl-related PTC; nearly all to RET/PTC1 or RET/PTC3, resulting from paracentric inversion of chromosome 10. Other less common variants, usually forming as a result of interchromosomal translocation, occur in an extremely limited number of cases. Most often such translocations have been found in radiation-induced PTC. Here, we report the presence of RET oncogene amplification, a new type of RET cytogenetic alteration, in human thyroid cancers. RET amplification was correlated with radiation-associated, high-grade malignant potency, and p53 overexpression, suggesting genomic instability. We also examined p53-binding protein 1 (53BP1) expressions with immunostaining to clarify the presence of genomic instability in thyroid cancers. In thyroid tumors, the level of 53BP1-focus formation was associated with malignant transformation and high-grade malignant potency as well as RET amplification. RET amplification might be induced by a high level of genomic instability with progression of thyroid carcinogenesis and, subsequently, be associated with radiation-induced and/or high-grade malignant cases.

Kinase Domain Mutants of Bcr-Abl Exhibit Altered Transformation Potency, Kinase Activity, and Substrate Utilization, Irrespective of Sensitivity to Imatinib.

Background: Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired resistance. Mutations of the ATP binding p-loop are associated with a poor prognosis compared to other mutations, irrespective of imatinib sensitivity. To elucidate the underlying mechanisms we compared five KD mutants in biological and biochemical assays.Methods and Results: In competition experiments using Ba/F3 cells expressing native or KD mutated Bcr-Abl, proliferation rates of Y253F and E255K were highest, followed by T315I, H396P, native Bcr-Abl and M351T. Similar results were seen in semi-quantitative B-cell transformation assays, where Y253F and E255K showed the most rapid outgrowth, while M351T was slowest. In contrast, when compared to native Bcr-Abl,in myeloid colony formation assays Y253F and E255K showed comparable and M351T and H396P reduced transformation potency, while the potency of T315I was reduced only in the absence of cytokines. The approximate consensus ranking of transformation potency from the different in vitro assays was Y253F > E255K > native Bcr-Abl > T315I > H396P > M351T.These differences were partially reflected in a murine leukemia model, where we observed trends for longer survival in recipients of H396P infected marrow and shorter survival in recipients of E255K. Next, we assayed kinetic properties of the purified KDs and recombinant full length Bcr-Abl in in vitro kinase assays. For the isolated KD, the Kcat/Km of the Y253F mutant was increased over native Bcr-Abl, E255K was similar and the other mutants were slightly (H396P) or significantly (M351T and T315I) reduced. Results with full-length proteins were similar for Y253F, T315I and M351T, while Kcat/Km for E255K was slightly reduced and for H396P slightly increased. Thus, the transformation potency of Y253F, M351T and H396P correlated with intrinsic kinase activity, while the biological activity of E255K and T315I was greater than would be predicted based on kinase assay results. We hypothesized that differential activation of signaling pathways may further modulate transformation potency. Indeed, phosphotyrosine proteome analysis by mass spectrometry revealed differential phosphorylation among the mutants, consistent with altered substrate specificity and pathway activation. For example, phosphorylation of Y1021 of SHIP1 was detected in Ba/F3 cells expressing the M351T and H396P mutant but not in cells expressing other KD mutants or native Bcr-Abl. This was confirmed by immunoblot analysis. Additional experiments showed significant differences by genome-wide microarray expression profiling, consistent with differential activation of signaling pathways.Conclusion: Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, the net results of which is gain or loss of function variants. Drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.