Bortezomib decreases Rb phosphorylation and induces caspase-dependent apoptosis in Imatinib-sensitive and -resistant Bcr-Abl1-expressing cells.

The use of c-abl-specific inhibitors such as Imatinib (IM) or Dasatinib has revolutionized the treatment of chronic myeloid leukemia (CML). However, a significant percentage of patients become resistant to IM. In this report, we have analyzed the possibility of using the proteasome as a molecular target in CML. Our results show that cells that express Bcr-Abl1 are more sensitive to the inhibition of the proteasome with Bortezomib (Btz) than control cells. This treatment reduces the proliferation of Bcr-Abl1-expressing cells, by inactivating NF-kappaB2 and decreasing the phosphorylation of Rb, eventually leading to an increase in caspase-dependent apoptosis. Furthermore, we show that Btz also induces cell-cycle arrest and apoptosis in cells expressing Bcr-Abl1 mutants that are resistant to IM. These results unravel a new molecular target of Btz, that is the Rb pathway, and open new possibilities in the treatment of CML especially for patients that become resistant to IM because of the presence of the T315I mutation.

Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia.

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.

Antiapoptotic protein Bcl-x(L) is up-regulated during megakaryocytic differentiation of CD34(+) progenitors but is absent from senescent megakaryocytes.

OBJECTIVE: The expression of Bcl-x(L) has been shown to be regulated during the maturation process of different hematopoietic cell lineages (i.e., erythroid cells, neutrophils, monocytes/macrophages). In the present study, we examined the expression of Bcl-x(L) in megakaryocytes derived from CD34(+) progenitors and in the megakaryoblastic cell line UT7. MATERIALS AND METHODS: Expression of Bcl-x(L) was analyzed in CD41(+) cells cultured in the presence of thrombopoietin and in UT7 cells treated with phorbol diester by Western blot, flow cytometry, and immunocytochemistry analysis. Apoptosis was determined at different culture times by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and propidium iodide uptake. RESULTS: Bcl-x(L) but not Bcl-2 was up-regulated in the megakaryocytic population (CD41(+)) during the first 15 days of culture, which was consistent with the pattern of Bcl-x(L) expression in UT7 cells differentiated to megakaryocytes by incubation with phorbol diester. However, by day 20 of culture, the levels of Bcl-x(L) in CD41(+) cells were greatly reduced, and this expression pattern was accompanied by an increase in the number of apoptotic cells. At this culture time, we detected the presence of cytoplasmic fragments resembling proplatelets with prominent Bcl-x immunostaining, most likely due to the Bcl-x(L) isoform, in close proximity to Bcl-x(-) senescent megakaryocytes. The presence of Bcl-x(L) but not of Bcl-2 in platelets was confirmed by Western blot analysis. CONCLUSION: Although little is known regarding the functional significance of survival proteins within the megakaryocytic compartment, the changes in the Bcl-x(L) expression pattern observed in UT7 and CD41(+) cells may play a role in the survival of developing megakaryocytes and the lifespan of mature platelets.

Electrothermal atomic absorption spectrometric diagnosis of familial hypercholesterolemia.

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies.

Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL.

Bcr-Abl-expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl-expressing cell lines and CD34(+) cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.

A rapid procedure suitable to assess quantitatively the endocytosis of colloidal gold and its conjugates in cultured cells.

We measured the endocytic uptake of low-density lipoproteins (LDLs) conjugated to colloidal gold in cultured cells, either by counting gold particles on electron micrographs or by inductively coupled plasma (ICP) mass spectrometry (MS). Both procedures are comparable but the latter requires a considerably shorter time and allows analysis of a much larger sample. In addition, ICP MS, compared to alternative radioactive or fluorescent procedures, offers the major advantage of using the same probe to quantify the endocytic uptake and to follow it by electron microscopy. Therefore, ICP MS analysis provides an easy, rapid, and sensitive quantification of endocytosis that complements the electron microscopic studies.

Use of inductively coupled plasma-mass spectrometry for the quantitation of the binding and uptake of colloidal gold-low-density lipoprotein conjugates by cultured cells.

The binding and endocytic uptake of low-density lipoprotein (LDL) particles by cells, transiently or permanently transfected with the human LDL receptor cDNA, was investigated, under different situations, using colloidal gold-LDL conjugates. The amount of gold associated with the various cells, which bind and internalize LDL to different extents, was estimated by inductively coupled plasma-MS. In all cases, the existing differences in LDL binding and uptake were clearly detectable with this procedure. We conclude, therefore, that inductively coupled plasma-MS provides an appropriate assay system for the rapid quantitation of these processes. This procedure also recognizes differences in LDL receptor expression in human lymphocytes and, therefore, it could be of value for the differential diagnosis of LDL receptor defects in familial hypercholesterolemia in various cell types. In addition, this easily performed methodology can also be applied to a variety of other problems requiring quantitation of colloidal gold associated with cells.