Low dose out-of-field radiotherapy, part 2: Calculating the mean photon energy values for the out-of-field photon energy spectrum from scattered radiation using Monte Carlo methods.

PURPOSE: During radiotherapy, leakage from the machine head and collimator expose patients to out-of-field irradiation doses, which may cause secondary cancers. To quantify the risks of secondary cancers due to out-of-field doses, it is first necessary to measure these doses. Since most dosimeters are energy-dependent, it is essential to first determine the type of photon energy spectrum in the out-of-field area. The aim of this study was to determine the mean photon energy values for the out-of-field photon energy spectrum for a 6 MV photon beam using the GEANT 4-Monte Carlo method. MATERIAL AND METHODS: A specially-designed large water phantom was simulated with a static field at gantry 0°. The source-to-surface distance was 92cm for an open field size of 10×10cm2. The photon energy spectra were calculated at five unique positions (at depths of 0.5, 1.6, 4, 6, 8, and 10cm) along the central beam axis and at six different off-axis distances. RESULTS: Monte Carlo simulations showed that mean radiation energy levels drop rapidly beyond the edge of the 6 MV photon beam field: at a distance of 10cm, the mean energy level is close to 0.3MeV versus 1.5MeV at the central beam axis. In some cases, the energy level actually increased even as the distance from the field edge increased: at a depth of 1.6cm and 15cm off-axis, the mean energy level was 0.205MeV versus 0.252MeV at 20cm off-axis. CONCLUSION: The out-of-field energy spectra and dose distribution data obtained in this study with Monte Carlo methods can be used to calibrate dosimeters to measure out-of-field radiation from 6MV photons.

Low dose out-of-field radiotherapy, part 1: Measurement of scattered doses.

PURPOSE: To measure out-of-field doses in a phantom model to better quantify this radiation. MATERIAL AND METHODS: The individual contribution of photons and neutrons to the total out-of-field dose for 6 MV and 20 MV photons at open beam were measured in a purpose-designed water phantom. Radiation doses were measured at seven separate points (P1-P7) in the phantom with thermoluminescent detectors (TLD 100, 600, and 700) and GAFchromic™ EBT films. RESULTS: At a prescribed dose of 75Gy to the isocentre, the photon dose level in the close-to-field area (P2) ranged from 2.0-2.5Gy for 6 MV and 1.5-2.0Gy for 20 MV; the total out-of-field doses at P2 and P7, respectively, were estimated to be as follows: for 6 MV: TLD 100 (<3.23% and<0.14%); radiochromic film (<2.52% and <0.03%); and for 20 MV: TLD 100 (<2.94% and <0.78%); TLD 700 (<2.02% and <0.14%); and radiochromic film (<1.73% and <0.01%). Although the dose decreased rapidly as the distance from the central beam axis increased, even distant doses could be as high as several centigrays. The neutron dose for 20 MV photons at a distance of 25cm from the isocentre was 4.0mSv/Gy. CONCLUSION: Our results show that in the close-to-field area, the dose level could be as high as 1.5Gy assuming a prescribed dose of 75Gy to the isocentre. By contrast, the doses delivered to more distant areas from the planning target volume were much lower (centigrays). These findings show that both 6 MV and 20 MV photons could produce dosimetrically important dose levels outside of the field. The data reported here may be of value to study the potential impact of even very low doses of radiation on human tissues.

Low dose out-of-field radiotherapy, part 3: Qualitative and quantitative impact of scattered out-of-field radiation on MDA-MB-231 cell lines.

PURPOSE: Patients who undergo external beam radiotherapy are at risk of developing second tumours due to scattered radiation outside the path of the primary beam. The aim of this study was to experimentally determine the in vitro radiobiological effects of scattered radiation in cells located outside the primary photon beam and to compare this to the effects that occur in cells inside the primary beam. The comparison was performed by assessing cell viability, DNA damage, and apoptosis. MATERIAL AND METHODS: Cells from the human breast cancer line MDA-MB-231 were inserted in a water phantom and irradiated at varying doses (1.5, 2.0, 2.5, and 3.0Gy). The cells were placed at two geometrical points: in the central beam axis and at 10cm out-of-field. The dose was constant in both geometrical points. Survival fraction, number of DNA double strand-breaks, and cleaved poly-(ADP-ribose) polymerase (PARP) levels were determined by clonogenic assay and flow cytometry. RESULTS: A slight, non-significant decrease of 3 to 5% in cell survival fraction was observed in cells irradiated outside the primary field. The number of PARP-positive cells and DNA double strand-breaks both increased after out-of-field irradiation. CONCLUSION: Scattered irradiation appears to induce an in vitro biological response on out-of-field cells that is stronger than the effect of primary radiation on in-field cells, independent of the bystander effect. These findings suggest that the biological response of healthy tissues outside the primary beam might be higher than previously believed.

Impact of the Intra- and Inter-observer Variability in the Delineation of Parotid Glands on the Dose Calculation During Head and Neck Helical Tomotherapy.

The intra- and inter-observer variability in delineation of the parotids on the kilo-voltage computed tomography (kVCT) and mega-voltage computed tomography (MVCT) were examined to establish their impact on the dose calculation during adaptive head and neck helical tomotherapy (HT). Three observers delineated left and right parotids for ten randomly selected patients with oropharynx cancer treated on HT. The pre-treatment kVCT and the MVCT from the first fraction of irradiation were selected to delineation. The delineation procedure was repeated three times by each observer. The parotids were delineated according to the institutional protocol. The analyses included intra-observer reproducibility and inter-structure, -observer and -modality variability of the volume and dose. The differences between the left and right parotid outlines were not statistically significant (p > 0.3). The reproducibility of the delineation was confirmed for each observer on the kVCT (p > 0.2) and on the MVCT (p > 0.1). The inter-observer variability of the outlines was significant (p < 0.001) as well as the inter-modality variability (p < 0.006). The parotids delineated on the MVCT were 10% smaller than on the kVCT. The inter-observer variability of the parotids delineation did not affect the average dose (p = 0.096 on the kVCT and p = 0.176 on the MVCT). The dose calculated on the MVCT was higher by 3.3% than dose from the kVCT (p = 0.009). Usage of the institutional protocols for the parotids delineation reduces intra-observer variability and increases reproducibility of the outlines. These protocols do not eliminate delineation differences between the observers, but these differences are not clinically significant and do not affect average doses in the parotids. The volumes of the parotids delineated on the MVCT are smaller than on the kVCT, which affects the differences in the calculated doses.

A dosimetric comparison of IMRT versus helical tomotherapy for brain tumors.

BACKGROUND AND PURPOSE: Helical tomotherapy (HT) can deliver highly conformal, uniform doses to the target volume. However, HT can only be delivered in a coplanar mode. The purpose of this study was to perform a dosimetric comparison of HT versus coplanar (cIMRT) and non-coplanar (n-cIMRT) beam arrangements on a conventional linear accelerator in a diverse group of brain tumors. MATERIALS AND METHODS: A total of 45 treatment plans were calculated retrospectively for 15 cases. For each case, 3 different delivery techniques (n-cIMRT, cIMRT and HT) were used. The treatment plans were compared using the parameters of the target coverage (conformity index; CI) and homogeneity (HI) for the planning target volume (PTV) and the maximum and mean doses for organs at risk (OARs). RESULTS: Median HI and CI were the best for HT plans and the worst for cIMRT. The largest reduction of maximum dose for lenses and mean dose for both eyes was achieved for n-cIMRT plans. Mean dose for chiasm and the ipsilateral optic nerve were the lowest for HT. The contralateral optic nerve was most spared with n-cIMRT. For D1% in the brain stem, there was no significant difference between HT and the IMRT plans. CONCLUSIONS: Both HT and n-cIMRT are capable of producing conformal and homogeneous treatment plans with a good sparing of OARs. However, due to the non-coplanar capabilities of IMRT, n-cIMRT led to a superior dose reduction to the lenses.

Impact of the Intra- and Inter-observer Variability in the Delineation of Parotid Glands on the Dose Calculation During Head and Neck Helical Tomotherapy.

The intra- and inter-observer variability in delineation of the parotids on the kilo-voltage computed tomography (kVCT) and mega-voltage computed tomography (MVCT) were examined to establish their impact on the dose calculation during adaptive head and neck helical tomotherapy (HT). Three observers delineated left and right parotids for ten randomly selected patients with oropharynx cancer treated on HT. The pre-treatment kVCT and the MVCT from the first fraction of irradiation were selected to delineation. The delineation procedure was repeated three times by each observer. The parotids were delineated according to the institutional protocol. The analyses included intra-observer reproducibility and inter-structure, -observer and -modality variability of the volume and dose. The differences between the left and right parotid outlines were not statistically significant (p > 0.3). The reproducibility of the delineation was confirmed for each observer on the kVCT (p > 0.2) and on the MVCT (p > 0.1). The inter-observer variability of the outlines was significant (p < 0.001) as well as the inter-modality variability (p < 0.006). The parotids delineated on the MVCT were 10% smaller than on the kVCT. The inter-observer variability of the parotids delineation did not affect the average dose (p = 0.096 on the kVCT and p = 0.176 on the MVCT). The dose calculated on the MVCT was higher by 3.3% than dose from the kVCT (p = 0.009). Usage of the institutional protocols for the parotids delineation reduces intra-observer variability and increases reproducibility of the outlines. These protocols do not eliminate delineation differences between the observers, but these differences are not clinically significant and do not affect average doses in the parotids. The volumes of the parotids delineated on the MVCT are smaller than on the kVCT, which affects the differences in the calculated doses.

Tomotherapy: implications on daily workload and scheduling patients based on three years' institutional experience.

Helical tomotherapy (HT) was introduced at the Greater Poland Cancer Centre (GPCC) in April 2009. Retrospective analysis included data from the treatments performed for the first 656 patients treated with HT between May 2009 and May 2012 at the GPCC. In order to evaluate the implications on daily workload and scheduling of patients, stepwise regression and time analysis for each component of the overall treatment time, such as positioning, imaging, registration, and irradiation were performed. A detailed analysis included: (1) learning curves and optimized time needed for positioning and registration; (2) relation between irradiation time and parameters used for plan creation; and (3) average time of daily imaging. The irradiation component has the highest influence on the overall treatment time (R = 0.911). The lowest influence was observed for the imaging (R = 0.670). The learning curve for positioning was 7 months while the reduction of the average daily time needed for registration was observed even after two years. The irradiation time strongly depends on the planning parameters. Changing the pitch from 0.215 to 0.287 for pelvic cancer cases decreased the average daily beam-on time per patient by about 2 minutes. Similar changes for head and neck reduced this time by 1.3 minutes. The limitation in the usage of 1 cm field width only for complex cases, lower than 10 cm in the cranio-caudal direction, reduced the beam-on time per patient by 2 minutes. The average overall treatment time decreased from 21.5 minutes per patient in the first year of the HT usage to 13.8 minutes per patient in current practice. Our current practice shows that for a group of patients including mainly those with pelvis and head and neck cancers, the HT treatment takes approximately 15 minutes per patient allowing 40 patients to be treated within 10 hours.

Optimization of treatment planning parameters used in tomotherapy for prostate cancer patients.

BACKGROUND AND PURPOSE: Tomotherapy treatment planning depends on parameters that are not used conventionally such as: field width (FW), pitch factor (PF) and modulation factor (MF). The aim of this study is to analyze the relationship between these parameters and their influence on the quality of treatment plans and beam-on time. MATERIAL AND METHODS: Ten prostate cancer patients were included in the study. For each patient, two cases of irradiation were considered depending on the target volume: PTV1 included the prostate gland, seminal vesicles, pelvic lymph nodes and a 1 cm margin, whereas PTV2 included only the prostate gland with a 1 cm margin. For each patient and each case of irradiation (PTV1 and PTV2) 8 treatment plans were created - all consisted of a different combination of planning parameters (FW = 1.05, 2.5, 5 cm; PF = 0.107, 0.215, 0.43; MF = 1.5, 2.5, 3.5). Default values used in this study were FW = 2.5 cm, PF = 0.215 and MF = 2.5. Hence, for plans with different FWs, parameters of PF and MF were 0.215 and 2.5, respectively; for different PFs, FW and MF were 2.5 and 2.5, respectively; finally for different MFs, FW and PF were 2.5 and 0.215, respectively. The reference plan was optimized for FW = 1.05 cm, PF = 0.107 and MF = 3.5, which was assumed to result in the best dose distribution and the longest treatment time. As a result, 160 plans were created. Each plan was analyzed for dose distribution and execution time. RESULTS AND CONCLUSION: : Treatment plans with FW of 5 cm resulted in the shortest execution time compromising the dose distribution. Moreover, the dose fall off in the longitudinal direction was not sharp. FW of 1.05 cm and PF of 0.107 were not recommended for routine prostate plans due to long execution time, which was 3 times longer than for plans with FW = 5 cm. There was no substantial decrease of irradiation time when PF was increased from 0.215 to 0.43 for both cases (PTV1 and PTV2); however, the dose distribution was slightly compromised. Finally, decreasing MF from 2.5 to 1.5 was useless because it did not change the beam-on time; however, it did remarkably decrease the dose distribution. Nevertheless, increasing MF up to 3.5 could be considered. The lowest EUD for the rectum and intestines, could be observed for PF = 0.107. For the other plans the differences were rather small (the EUD was almost the same). By reducing PF from 0.43 to 0.107 or FW from 5 to 1.05 the EUD for bladder (in PTV1 case) decreased by 3.13% and 2.60%. When PTV2 was a target volume, the EUD for bladder decreased by 4.54% and 3.43% when FW was changed from 5 to 1.05 and MF from 1.5 to 3.5, respectively. For optimal balance between beam-on time and dose distribution in OARs for routine patients, the authors would suggest to use: FW = 2.5, PF = 0.215 and MF = 2.5.

BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance.

RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.

Role of signal transducer and activator of transcription 5 in nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in anaplastic large-cell lymphoma, encodes a M(r) 75,000 hybrid protein that containsthe amino-terminal portion of the nucleolar phosphoprotein nucleophosmin(NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocation. Our studies show that NPM/ALK, similar to other members of this family, activates signal transducer and activator of transcription 5 (STAT5) and that this activation is essential for lymphomagenesis. NPM/ALK-mediated activation of STAT5 was demonstrated by detection of: (a) constitutive tyrosine phosphorylation and enhanced DNA binding ability of STAT5 in NPM/ALK-transformed cells; and (b) NPM/ALK-dependent stimulation of STAT5-mediated transactivation of the beta-casein promoter. Retroviral infection of NPM/ALK+ cells with a dominant-negative STAT5B mutant (STAT5-DNM) inhibited the antiapoptotic activity of NPM/ALK in growth factor and serum-free medium. In addition, STAT5-DNM inhibited proliferation and diminished the clonogenic properties of NPM/ALK-positive cells. Finally, SCID mice injected with NPM/ALK+ cells infected with a virus carrying STAT5-DNM survived significantly longer than mice inoculated with NPM/ALK+ cells infected with the empty virus. Necropsy identified a widespread ALK+ lymphoma in lymph nodes and liver of the affected animals. Together, our data indicate that NPM/ALK-induced activation of STAT5 may play an important role in NPM/ALK-mediated lymphomagenesis.

Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (delta p85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.

Progressive changes in the leukemogenic signaling in BCR/ABL-transformed cells.

Our previous study indicated that BCR/ABL SH2 domain and BCR/ABL SH3 domain+SH2 domain complex are required for immediate activation of the phosphatidylinositol-3 kinase PI-3k)--> Akt serine/threonine kinase pathway and of the signal transducer and activator of transcription 5 (STAT5), respectively, in hematopoietic cells. We show here that the defect in activation of PI-3k/Akt by BCR/ABL DeltaSH2 mutant (SH2 domain deleted) and of STAT5 by BCR/ABL DeltaSH3+DeltaSH2 mutant (SH3 and SH2 domains deleted) is not permanent and both Akt and STAT5 could be 're-activated' by in vitro culture. This phenomenon was responsible for increased resistance to apoptosis, growth factor-independent proliferation and leukemogenesis in SCID mice. Incubation of cells with BCR/ABL tyrosine kinase inhibitor STI571 abrogated the 're-activation' of Akt or STAT5 by BCR/ABL SH3+SH2 mutants in some clones, in the others Akt and STAT5 activation became independent on BCR/ABL kinase activity. The immediate upstream activators of Akt and STAT5 such as PI-3k and Jak-2 were also activated. In addition, the common beta subunit of IL-3/IL-5/GM-CSF receptor was tyrosine phosphorylated in the clones in which 're-activation' was dependent on the BCR/ABL kinase activity. These results suggested that 're-activation' of Akt and STAT5, in the absence of functional BCR/ABL SH3+SH2 domains, may be achieved by two different mechanisms: (i) BCR/ABL kinase-dependent activation of alternative pathway(s) and (ii) additional genetic changes stimulating Akt and STAT5 independently of BCR/ABL. Oncogene (2000) 19, 4117 - 4124

Activation of mitochondrial Raf-1 is involved in the antiapoptotic effects of Akt.

The Akt serine/threonine kinase is required for the survival of many cell types and for transformation of hematopoietic cells by the BCR/ABL oncogenic tyrosine kinase. Analysis of the potential mechanisms whereby Akt promotes survival of hematopoietic cells revealed that it induced the activity of plasma membrane and mitochondrial Raf-1 in a Ras-independent, but PKC-dependent manner. Inhibition of plasma membrane Raf-1-dependent mitogen-activated protein kinase activity had no effect on the enhanced survival of cells expressing Akt. By contrast, suppression of mitochondrial Raf-1 enzymatic activity by expression of a mitochondria-targeted Raf-1 dominant-negative mutant rendered Akt-expressing cells susceptible to apoptosis induced by growth factor deprivation and was accompanied by inhibition of BAD, but not mitogen-activated protein kinase, phosphorylation. Together, these data indicate that PKC-dependent activation of Raf-1 plays an important role in Akt-dependent antiapoptotic effects.

Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis.

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor-dependent myeloid precursor cells, STAT5 activation-deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor-independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor-independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation-defective BCR/ABL SH3+SH2 mutants to induce growth factor-independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor-independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

BCR/ABL-mediated leukemogenesis requires the activity of the small GTP-binding protein Rac.

The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2. We show here that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retained the resistance to growth factor deprivation-induced apoptosis but showed a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants were capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants was markedly prolonged as compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis.

The SH3 domain contributes to BCR/ABL-dependent leukemogenesis in vivo: role in adhesion, invasion, and homing.

To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (delta SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. delta SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of delta SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing delta SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of delta SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of alpha 2 integrin by delta SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/membrane fraction, delta SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway.

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3-kinase (PI-3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI-3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI-3k activation failed to stimulate Akt kinase, a recently identified PI-3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI-3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI-3k specific inhibitor at low concentrations, suppressed BCR/ABL-dependent colony formation of murine marrow cells, and that a kinase-deficient Akt mutant with dominant-negative activity inhibited BCR/ABL-dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation-defective SH2 domain BCR/ABL mutants to induce growth factor-independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c-Myc and Bcl-2; in contrast, expression of a constitutively active Akt mutant induced Bcl-2 and c-Myc expression, and stimulated the transcription activation function of c-Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI-3k activation and document the essential role of the PI-3k/Akt pathway in BCR/ABL leukemogenesis.

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.