Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Postoperative insulin secretion is decreased in patients with preoperative insulin resistance.

BACKGROUND: Postoperative hyperglycemia is associated with increased rate of surgical site infection, renal failure, and cardiovascular events. The study of insulin sensitivity state before surgery could help in treating postoperative hyperglycemia and preventing iatrogenic hypoglycemia. We studied the postoperative insulin secretion in patients who have a low insulin sensitivity (IR) before surgery compared to patients with normal preoperative insulin sensitivity (IS). MATERIALS AND METHODS: Forty-two consecutive patients, undergoing abdominal surgery, underwent preoperative sequential hyperglycemic-euglycemic clamp (SHEC) in order to measure insulin secretion and to screen patients with low insulin sensitivity (IR) or with normal insulin sensitivity (IS). Patients had been randomized to receive either general anesthesia with epidural or PCA. RESULTS: Postoperative insulin secretion in IR patients is decreased compared to IS (P = 0.059) and to IR before surgery regardless to the type of analgesia (P < 0.001). In the IS group, postoperative insulin secretion depends on type of analgesia. It is increased when using PCA and decreased when using epidural (P < 0.05). Blood glucose increased after surgery in both IS an IR (P < 0.001). Patients with preoperative insulin resistance had a higher glycemia before and after surgery (P < 0.001). Blood glucose levels were comparable between PCA and epidural patients (P = 0.450). CONCLUSION: Insulin secretion is reduced in IR regardless the type of anesthesia. PCA increases insulin secretion, whereas epidural decreases it in patients with normal insulin sensitivity. These findings implicate that after surgery insulin administration is advisable in patients with preoperative insulin resistance while it should be given cautiously in those with normal preoperative insulin sensitivity.

A phase II study of a human anti-PDGFRα monoclonal antibody (olaratumab, IMC-3G3) in previously treated patients with metastatic gastrointestinal stromal tumors.

Background: This study evaluated tumor response to olaratumab (an anti-PDGFRα monoclonal antibody) in previously treated patients with metastatic gastrointestinal stromal tumor (GIST) with or without PDGFRα mutations (cohorts 1 and 2, respectively). Patients and methods: Patients received olaratumab 20 mg/kg intravenously every 14 days until disease progression, death, or intolerable toxicity occurred. Outcome measures were 12-week tumor response, progression-free survival (PFS), overall survival (OS), and safety. Results: Of 30 patients enrolled, 21 patients received ≥1 dose of olaratumab. In the evaluable population (cohort 1, n = 6; cohort 2, n = 14), no complete response (CR) or partial response (PR) was observed. Stable disease (SD) was observed in 3 patients (50.0%) in cohort 1 and 2 patients (14.3%) in cohort 2. Progressive disease (PD) was observed in 3 patients (50.0%) in cohort 1 and 12 patients (85.7%) in cohort 2. The 12-week clinical benefit rate (CR + PR + SD) (90% CI) was 50.0% (15.3-84.7%) in cohort 1 and 14.3% (2.6-38.5%) in cohort 2. SD lasted beyond 12 weeks in 5 patients (cohort 1, n = 3; cohort 2, n = 2). Median PFS (90% CI) was 32.1 (5.0-35.9) weeks in cohort 1 and 6.1 (5.7-6.3) weeks in cohort 2. Median OS was not reached in cohort 1 and was 24.9 (14.4-49.1) weeks in cohort 2. All patients in cohort 1 and 9 (64.3%) in cohort 2 experienced an olaratumab-related adverse event (AE), most commonly fatigue (38.1%), nausea (19.0%), and peripheral edema (14.3%). Two grade ≥3 olaratumab-related events were reported (cohort 1, syncope; cohort 2, hypertension). Conclusions: Olaratumab had an acceptable AE profile in patients with GIST. While there was no apparent effect on PFS in patients without PDGFRα mutations, patients with PDGFRα-mutant GIST (all with D842V mutations) treated with olaratumab had longer disease control compared with historical data for this genotype. ClinicalTrials.gov Identifier: NCT01316263.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3-dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)-like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)-treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non- 5-FU-treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.

Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein.

The RING-finger domain is a novel zinc-binding Cys-His protein motif found in a growing number of proteins involved in signal transduction, ubiquitination, gene transcription, differentiation, and morphogenesis. We describe a novel muscle-specific RING-finger protein (MURF) expressed specifically in cardiac and skeletal muscle cells throughout pre- and postnatal mouse development. MURF belongs to the RING-B-box-coiled-coil subclass of RING-finger proteins, characterized by an NH(2)-terminal RING-finger followed by a zinc-finger domain (B-box) and a leucine-rich coiled-coil domain. Expression of MURF is required for skeletal myoblast differentiation and myotube fusion. The leucine-rich coiled-coil domain of MURF mediates association with microtubules, whereas the RING-finger domain is required for microtubule stabilization and an additional region is required for homo-oligomerization. Expression of MURF establishes a cellular microtubule network that is resistant to microtubule depolymerization induced by alkaloids, cold and calcium. These results identify MURF as a myogenic regulator of the microtubule network of striated muscle cells and reveal a link between microtubule organization and myogenesis.

Fatal myeloproliferation, induced in mice by TEL/PDGFbetaR expression, depends on PDGFbetaR tyrosines 579/581.

The t(5;12)(q33;p13) translocation associated with chronic myelomonocytic leukemia (CMML) generates a TEL/PDGFbetaR fusion gene. Here, we used a murine bone marrow transplant (BMT) assay to test the transforming properties of TEL/PDGFbetaR in vivo. TEL/PDGFbetaR, introduced into whole bone marrow by retroviral transduction, caused a rapidly fatal myeloproliferative disease that closely recapitulated human CMML. TEL/PDGFbetaR transplanted mice developed leukocytosis with Gr-1(+) granulocytes, splenomegaly, evidence of extramedullary hematopoiesis, and bone marrow fibrosis, but no lymphoproliferative disease. We assayed mutant forms of the TEL/PDGFbetaR fusion protein - including 8 tyrosine to phenylalanine substitutions at phosphorylated PDGFbetaR sites to which various SH2 domain-containing signaling intermediates bind - for ability to transform hematopoietic cells. All of the phenylalanine (F-) mutants tested conferred IL-3-independence to a cultured murine hematopoietic cell line, but, in the BMT assay, different F-mutants displayed distinct transforming properties. In transplanted animals, tyrosines 579/581 proved critical for the development of myeloproliferative phenotype. F-mutants with these residues mutated showed no sign of myeloproliferation but instead developed T-cell lymphomas. In summary, TEL/PDGFbetaR is necessary and sufficient to induce a myeloproliferative disease in a murine BMT model, and PDGFbetaR residues Y579/581 are required for this phenotype.

A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.

Drainage of pleural effusion improves diaphragmatic function in mechanically ventilated patients.

BACKGROUND: Pleural effusion adversely affects the pressuregenerating capacity of the diaphragm. It uncouples the lung and chest wall, which may result in diaphragmatic dysfunction. Information on the effects of effusion drainage on diaphragmatic function is limited, but several studies report relief of dyspnoea after drainage, which was attributed to improved diaphragmatic mechanics, even if this issue was never formally addressed. OBJECTIVE: To investigate the effect of drainage of unilateral pleural effusion on diaphragmatic function. DESIGN, SETTING AND PATIENTS: In a prospective twostep protocol (at baseline and after drainage of effusion), we conducted a spontaneous breathing trial in fourteen critically ill, mechanically ventilated patients undergoing pressure support ventilation. MAIN OUTCOME MEASURES: We used ultrasonography of the ipsilateral hemidiaphragm to evaluate and record respiratory displacement and thickening during tidal and maximal breathing efforts. We recorded and analysed airway pressures, respiratory system compliance, vital capacity, indices of respiratory effort and arterial blood gases. RESULTS: After drainage of the effusion, the respiratory rate decreased and tidal volume increased, but haemodynamic parameters were unaffected and oxygenation levels showed a non-significant increase. Drainage was associated with significant decreases in indices of respiratory drive and the maximal pressure generated by the respiratory muscles, as well as an increased compliance of the respiratory system. Diaphragmatic displacement and thickening significantly increased after drainage. We found there was a significant correlation between the volume of the effusion drained and the increase in tidal diaphragmatic thickening. CONCLUSIONS: Drainage of a unilateral pleural effusion during weaning from mechanical ventilation improves diaphragmatic contractile activity and respiratory system performance.

Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1-dependent transformation.

The chimeric gene EWS/FLI-1, the hallmark of the Ewing's sarcoma and primitive neuroectodermal tumor family, encodes a fusion protein with enhanced transcriptional activation properties and preserved recognition of canonical ETS binding sites. Although EWS/FLI-1 alters the expression of various genes, the precise mechanism by which EWS/FLI-1 acts as an oncogene remains to be defined. In this study we report that members of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1 and ERK2, are constitutively activated in NIH 3T3 cells expressing EWS/FLI-1. Interference with ERK activation by either highly specific inhibitors of MEK1 or a dominant negative ras mutant profoundly impaired the ability of EWS/FLI-1 to transform NIH3T3 cells to growth in semi-solid medium. An EWS/FLI-1 mutant defective in DNA-binding and transcriptional activation failed to activate ERK and was also defective in 3T3 cell transformation. Constitutive ERK activation was also evident in several human Ewing's sarcoma tumor-derived cell lines. Interestingly, cells expressing the type II EWS/FLI-1 fusion, recently demonstrated more potent in transcriptional activation, showed even greater MAPK activation than cells expressing the more common type I fusion. These results implicate ERK activation in EWS/FLI-1 transformation and suggest that this signaling pathway may be important in the pathogenesis of Ewing's sarcoma. Oncogene (2000) 19, 4523 - 4530.

Clonality analysis of childhood ALL in remission: no evidence of clonal hematopoiesis.

We studied 98 female patients in remission (2-240 months) from childhood ALL to determine the clonality status of their hematopoiesis. Thirty-one (31.6%) were heterozygous at the PGK locus for the BstX1 endonuclease restriction site, permitting X-linked clonality assays to be performed. Two patients were in relapse at the time of study and were excluded. We used the PGK-PCR clonality assay (PPCA) to analyze DNA from PMN and mononuclear cells of the remaining 29 female patients. All (29/29) patients demonstrated polyclonal hematopoiesis. These data show that remission from childhood ALL involves reestablishment of polyclonally derived hematopoiesis in all patients studied.

Bupropion in depression: a tri-center placebo-controlled study.

In a double-blind, placebo-controlled, variable-dose study of 59 hospitalized nonpsychotic depressed patients, bupropion was significantly (p less than .05 to less than .001) more effective than placebo on measures of depression, anxiety, and global improvement. Statistically significant drug-placebo differences appeared as early as day 5 of treatment and increased on subsequent assessments. In an evaluation of Baseline X Treatment interactions, bupropion was particularly more effective than placebo in those patients with more severe depression. Placebo and bupropion groups had similar frequencies and severity of side effects. Laboratory data showed minimal differences between the two treatments. The most common adverse experience was mild dry mouth (20% of patients). Compared to placebo, bupropion was found to be effective in the treatment of depression and to have a favorable safety profile.

Palliation of bronchogenic carcinoma with 198Au implantation using the fiberoptic bronchoscope.

The majority of cases of bronchogenic carcinoma remain incurable, and many of these patients require palliation of the effects of the tumor on the airway. We have developed a technique for implanting radioactive (198Au) seeds via the fiberoptic bronchoscope. We now retrospectively review the results obtained in 111 procedures in 54 patients. Response was assessed by improvement in symptoms, chest roentgenogram, or bronchoscopic appearance. Nineteen of 29 (66 percent) patients with occluding endobronchial lesions benefitted. Twenty of 22 (91 percent) with hemoptysis improved. All six patients with tracheal lesions benefitted. Two of six (33 percent) patients with nonoccluding endobronchial lesions responded. Complications directly related to the procedure were rarely of major consequence, although a single patient had an exsanguinating hemoptysis four days following the last of multiple implantations. The simplicity, relative safety, and potential wide availability coupled with low equipment costs would suggest an increasing role for this technique in the palliation of endobronchial neoplasms.

A rapid microprocedure for isolating RNA from multiple samples of human and rat brain.

In order to establish a routine procedure for isolating undegraded RNA from small amounts of rat and human brain tissue, several techniques were investigated. Initial studies demonstrated that undegraded RNA could not be reproducibly isolated from milligram amounts of brain tissue homogenized in an aqueous medium. Several isolation techniques utilizing tissue homogenization in the denaturing agent guanidinium chloride were compared. This method of homogenization, followed by sedimentation of RNA through cesium chloride, resulted in good yields of undegraded translationally active RNA. A maximum of 6 RNA samples could be processed simultaneously. In contrast, when homogenization in guanidinium chloride was followed by repeated guanidinium chloride-ethanol precipitations many samples could be processed simultaneously. The resulting RNA yields were low. The introduction of several modifications in the guanidinium chloride-ethanol precipitation technique resulted in a high yield of undegraded translationally active RNA. DNA was removed by two guanidinium-ethanol precipitations. Residual protein was digested with proteinase K. RNA was precipitated after extraction with phenol-chloroform-isoamyl alcohol. This refined procedure allows the recovery, in high yields, of translationally active undegraded RNA which is both DNA and protein free. Thirty-six samples can be processed in one day.

Drainage of pleural effusion in mechanically ventilated patients: time to measure chest wall compliance?

PURPOSE: Pleural effusion (PE) is commonly encountered in mechanically ventilated, critically ill patients and is generally addressed with evacuation or by fluid displacement using increased airway pressure (P(AW)). However, except when massive or infected, clear evidence is lacking to guide its management. The aim of this study was to investigate the effect of recruitment maneuvers and drainage of unilateral PE on respiratory mechanics, gas exchange, and lung volume. MATERIALS AND METHODS: Fifteen critically ill and mechanically ventilated patients with unilateral PE were enrolled. A 3-step protocol (baseline, recruitment, and effusion drainage) was applied to patients with more than 400 mL of PE, as estimated by chest ultrasound. Predefined subgroup analysis compared patients with normal vs reduced chest wall compliance (C(CW)). Esophageal and P(AW)s, respiratory system, lung and C(CW)s, arterial blood gases, and end-expiratory lung volumes were recorded. RESULTS: In the whole case mix, neither recruitment nor drainage improved gas exchange, lung volume, or tidal mechanics. When C(CW) was normal, recruitment improved lung compliance (81.9 [64.8-104.1] vs 103.7 [91.5-111.7] mL/cm H2O, P < .05), whereas drainage had no significant effect on total respiratory system mechanics or gas exchange, although it measurably increased lung volume (1717 vs 2150 mL, P < .05). In the setting of reduced C(CW), however, recruitment had no significant effect on total respiratory system mechanics or gas exchange, whereas pleural drainage improved respiratory system and C(CW)s as well as lung volume (42.7 [38.9-50.0] vs 47.0 [43.8-63.3], P < .05 and 97.4 [89.3-97.9] vs 126.7 [92.3-153.8] mL/cm H2O, P < .05 and 1580 vs 1750 mL, P < .05, respectively). CONCLUSIONS: Drainage of a moderate-sized effusion should not be routinely performed in unselected population of critically ill patients. We suggest that measurement of C(CW) may help in the decision-making process.

Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571.

The murine bone marrow retroviral transduction and transplantation model of chronic myelogenous leukemia (CML) imperfectly mimics human CML because the murine CML-like disease causes death of all animals from an overwhelming granulocytosis within 3 to 4 weeks. In this report, mice reconstituted with P210(BCR/ABL)-transduced bone marrow cells received posttransplantation therapy with either the tyrosine kinase inhibitor STI571 or placebo. Compared with the rapidly fatal leukemia of placebo-treated animals, 80% of the STI571-treated mice were alive on day 74, with marked improvement in peripheral white blood counts and splenomegaly. There was decreased tyrosine phosphorylation of STAT5, Shc, and Crk-L in leukemic cells from STI571-treated animals, consistent with STI571-mediated inhibition of the Bcr/Abl tyrosine kinase in vivo. In some STI571-treated animals Bcr/Abl messenger RNA and protein expression were markedly increased. In contrast to the polyclonal leukemia of placebo-treated mice, STI571-treated murine CML was generally oligoclonal, suggesting that STI571 eliminated or severely suppressed certain leukemic clones. None of the STI571-treated mice were cured of the CML-like myeloproliferative disorder, however, and STI571-treated murine CML was transplanted to secondary recipients with high efficiency. These results demonstrate the utility of this murine model of CML in the evaluation of novel therapeutic agents against Bcr/Abl-induced leukemias. This improved murine chronic-phase CML model may be a useful tool for the study of STI571 resistance, CML progression, and the anti-CML immune response.

The SH2 domain of P210BCR/ABL is not required for the transformation of hematopoietic factor-dependent cells.

Src-homology region 2 (SH2) domains, by binding to tyrosine-phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.