Activated abl oncogenes and apoptosis: differing responses of transformed myeloid progenitor cell lines

Activation of the c-abl protooncogene occurs during the generation of both the Abelson murine leukemia virus and the bcrabl fusion gene. To further dissect the biological properties of these proteins, we studied their effect on apoptosis. Using dimethyl sulfoxide (DMSO) to induce apoptosis in the murine myeloid progenitor cell line 32Dcl3, we examined the effect of expression of both v-abl and bcrabl transgenes on apoptosis. v-abl expressing 32Dcl3 cells are sensitive to apoptosis induction, similar to parental 32Dcl3 cells. In contrast, bcrabl expression 32Dcl3 cells are protected from the apoptotic stimulus resulting from DMSO exposure. Analyzing the expression patterns for Bcl- 2 and Bax, two proteins known to modulate the apoptotic response, we found a downregulation of Bcl-2 and enhanced expression of Bax in 32Dcl3 cells. In 32Dcl3/v-Abl cells, Bcl-2 expression remained constant while Bax was upregulated, whereas in 32Dcl3 cells expressing bcrabl, there was continuous expression of Bcl-2 at a level greater than observed in v-abl transformed cells. Taken together, our data demonstrate that although both activated abl gene products promote overlapping effects of some biological responses (i.e., factor- independent proliferation) they diverge in their effect on apoptotic signaling pathways.     

Hepatic imaging response to radioembolization with yttrium-90-labeled resin microspheres for tumor progression during systemic chemotherapy in patients with colorectal liver metastases

Background

To assess response and the impact of imaging artifacts following radioembolization with yttrium-90-labeled resin microspheres (⁹⁰Y-RE) based on the findings from a central independent review of patients with liver-dominant metastatic colorectal cancer (mCRC).

Methods

Patients with mCRC who received ⁹⁰Y-RE (SIR-Spheres^®; Sirtex Medical, Sydney, Australia) at nine US institutions between July 2002 and December 2011 were included in the analysis. Tumor response was assessed at baseline and 3 months using either the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 or 1.1. For each lesion, known artifacts affecting the interpretation of response (peri-tumoral edema and necrosis) were documented. Survivals (Kaplan-Meier analyses) were compared in responders [partial response (PR)] and non-responders [stable (SD) or progressive disease (PD)].

Results

Overall, 195 patients (mean age 62 years) received ⁹⁰Y-RE after a median of 2 (range, 1-6) lines of prior chemotherapy. Using RECIST 1.0 and RECIST 1.1, 7.6% and 6.9% of patients were partial responders, 47.3% and 48.1% had SD, and 55.0% and 55.0% PD, respectively. RECIST 1.0 and RECIST 1.1 showed excellent agreement {Kappa =0.915 [95% confidence interval (CI): 0.856-0.975]}. Peri-tumoral edema was documented in 32.8%, necrosis in 48.1% and both in 57.3% of cases (using RECIST 1.0). Although baseline characteristics were similar in responders and non-responders (P>0.05), responders survived significantly longer in an analysis according to RECIST 1.0: PR median (95% CI) 25.2 (range, 9.2-49.4) months vs. SD 15.8 (range, 9.3-21.1) months vs. PD 7.1 (range, 6.0-9.5) months (P<0.0001).

Conclusions

RECIST 1.0 and RECIST 1.1 imaging responses provide equivalent interpretations in the assessment of hepatic tumors following ⁹⁰Y-RE. Radiologic lesion responses at 3 months must be interpreted with caution due to the significant proportion of patients with peri-tumoral edema and necrosis, which may lead to an under-estimation of PR/SD. Nevertheless, 3-month radiologic responses were predictive of prolonged survival.     

Therapeutic angiogenesis

Abstract. Therapeutic angiogenesis improves tissue ischemia by supplementing and supporting the intrinsic process of angiogenesis. Besides direct administration of angiogenic growth factor proteins, injection of naked DNA, non-viral vectors and viral vector constructs carrying angiogenic genes have been used. A novel approach is to achieve therapeutic angiogenesis through cell mediated gene transfer. Genetically modulated cells carrying exogenous genes encoding for angiogenic factors and the cells with inherent ability to secrete angiogenic cytokines, such as bone marrow stem cells, embryonic stem cells and endothelial progenitor cells, have been used to achieve revascularization.This review discusses proof of the concept pre-clinical studies and phase-I/II human trials using VEGF, and cellular angiogenesis at length in the light of the literature and analyzes the problems and considerations of these approaches as a treatment strategy in the clinical perspective for the treatment of ischemic heart disease.     

Polymerase chain reaction-based diagnosis of del (5q) in acute myeloid leukemia and myelodysplastic syndrome identifies a minimal deletion interval

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.     

The xerocytosis of Hb SC disease

Patients with Hb SC disease were found to have microcytic and hyperchromic red cell indices despite mild reticulocytosis. Iron deficiency anemia was ruled out by the finding of normal serum ferritin levels. In order to determine whether the microcytosis was due to coexistent alpha-thalassemia, restriction endonuclease mapping was performed on genomic DNA extracted from peripheral blood leukocytes. Patients with Hb SC disease had microcytic indices despite the presence of a full complement of four alpha-genes (alpha alpha/alpha alpha), suggesting that the microcytosis may be due to cellular dehydration (or xerocytosis), since the mean corpuscular hemoglobin concentration in Hb SC disease patients was significantly higher than in controls. This possibility was investigated further by the determination of RBC cation content. RBC Na levels were similar in SC and normal red cells. Hb SC RBCs, however, had significantly reduced K levels. These findings show that RBC cation content, and thus cell water, is decreased in Hb SC disease. The decreased RBC K level in the presence of normal cellular Na concentration suggests selective K loss that is not due to inhibition of the Na K pump. Ouabain-insensitive K+ efflux was increased to four times normal in SC cells. Cell dehydration was confirmed by the demonstration of increased high-density RBCs on discontinuous Stractan density gradients and by osmotic gradient ektacytometry. Cellular dehydration and its sequelae were worse in CC erythrocytes and milder in AC cells than in Hb SC red cells. Taken together, these data indicate that in Hb SC disease the RBCs are severely dehydrated and typically microcytic and hyperchromic. Hb SC RBCs seem to be dehydrated due to selective K loss. These findings suggest a functional interrelationship between Hb SC, the red cell membrane, and cation regulation.     

ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding

DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA–protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the nonhydrolyzable ATP analog ADP-beryllium fluoride. Another ATP analog, ADP-aluminum fluoride, does not promote unwinding. The findings show that the energy from ATP hydrolysis is dispensable for strand separation. ATP binding, however, appears necessary. ATP hydrolysis is found to be required for fast enzyme release from the RNA and multiple substrate turnovers and thus for enzyme recycling.     

Amino acid substitutions in inherited albumin variants from Amerindian and Japanese populations.

We report an effort to determine the basis for the altered migration of seven inherited albumin variants detected by one-dimensional electrophoresis in population surveys involving tribal Amerindians and Japanese children. An amino acid substitution has thus far been determined for four of the variants. The randomness in the albumin polypeptide of these and the other sixteen independently ascertained amino acid substitutions of albumin and proalbumin thus far established was analyzed; the clustering of eight of these at two positions in the six-amino acid propeptide sequence seems noteworthy. By comparison with other proteins studied by electrophoresis, albumin exhibits "average" variability. It is a paradox that individuals who, for genetic reasons, lack albumin exhibit no obvious ill effects; yet, electrophoretic variants of albumin are no more numerous than are variants of proteins, the absence of which results in severe disease.