Gianluca Brusa

Endoplasmic reticulum stress initiates apoptotic death induced by STI571 inhibition of p210 bcr-abl tyrosine kinase

The endoplasmic reticulum (ER) is the site where proteins destined to either secretion or different subcellular compartments assemble and the major storage of intracellular Ca(2+). The ER stress resulting from a variety of toxic insults leads to apoptosis. Here, we showed that the apoptotic death triggered by STI571, an inhibitor of the p210 bcr-abl tyrosine kinase, in murine myeloid progenitors transducing the p210 bcr-abl tyrosine kinase of Chronic Myeloid Leukemia (CML) proceeds from ER stress. The Bcl-2 dowmodulation and inactivation induced by the binding to its antagonist: Bad, the release of caspase 12 from the ER membranes in its active form and of Ca(2+) from the ER pool addressed towards ER a sensor of STI571-induced pro-apoptotic signal.

P210 Bcr‐abl tyrosine kinase interaction with histone deacetylase 1 modifies histone H4 acetylation and chromatin structure of chronic myeloid leukaemia haematopoietic progenitors

The BCR‐ABL fusion gene, originating from the balanced (9;22) translocation, is the molecular hallmark and the causative event of chronic myeloid leukaemia (CML). The interactions of its p210 protein constitutively activated and improperly confined to the cytoplasm with multiple regulatory signals of cell cycle progression, apoptosis and self‐renewal, induce the illegitimate enlargement of clonal haematopoiesis and genetic instability that drives its progression towards the fully transformed phenotype of blast crisis. However, its effects on the basic transcription machinery and chromatin remodelling are unknown. Our study underscored histone H4 hyperacetylation associated with p210 tyrosine kinase in vitro and in vivo and its role in BCR‐ABL transcription. Histone H4 hyperacetylation proceeds, at least partly, from the ‘loss of function’ of histone deacetylase 1 protein, a critical component of Rb‐mediated transcriptional repression, in consequence of its cytoplasmatic compartmentalisation.

Acetylation of FOXO3a transcription factor in response to imatinib of chronic myeloid leukemia

Acetylation of FOXO3a transcription factor in response to imatinib of chronic myeloid leukemia

The p210BCR-ABL tyrosine kinase of chronic myeloid leukemia causes resistance to radio-induced apoptotic death by inhibiting the proapoptotic BAX gene.

The p210 BCR-ABL tyrosine kinase of chronic myeloid leukemia causes resistance to radio-induced apoptotic death by inhibiting the proapoptotic BAX gene

Expression of P21WAF1/CIP1/SID1 cyclin-dependent kinase inhibitor in hematopoietic progenitor cells

Abstract P21Waf1/Cip1/Sid1 is a critical component of biomolecular pathways leading to the G1 arrest evoked in response to DNA damage, growth arrest signals and differentiation commitment. It belongs to the Cip/Kip class of cyclin-dependent kinase inhibitors and is at least partly regulated by p53. P21Waf1/Cip1/Sid1 functional inactivation possibly resulting from mutations of the gene itself or, more likely, from p53 mutations may be critical for either the cell fate following DNA-damaging insults or clonal evolution toward malignancy. In the study presented here we describe a competitive polymerase chain reaction (PCR) strategy whose sensitivity and reproducibility enable us to attain a precise quantitation of p21Waf1/Cip1/Sid1 expression levels in hematopoietic progenitors, the cell compartment which mostly suffers from the side effects of genotoxic drugs in use for cancer cure. The strategy was set in the M07 factor-dependent hematopoietic progenitor cell line. We confirmed that its p21waf1/cip1/sid1 constitutive expression level is very low and up-modulated by DNA-damaging agents: ionizing radiations and ultraviolet light. Gene up-modulation resulted in checkpoint activation and, in particular, in a significant G1 arrest, required for either the repair of damaged DNA sequences or apoptotic cell death. Our competitive PCR strategy was further validated in CD34+ purified hematopoietic progenitors from healthy donors mobilized into the peripheral blood by granulocyte colony-stimulating factor and intended for allogeneic bone marrow transplantation. The constitutive p21WAF1/CIP1/SID1 expression levels, measured in three separate harvests, were very low and no significant differences were apparent. Our results support the use of a competitive PCR strategy as a useful tool for clinical purposes, to assess the individual biomolecular response of early hematopoietic progenitors to antiblastic drugs.

P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2

Purpose: To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS). Materials and methods: In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as ‘common’ deletion, by mean of a polimerase chain reaction (PCR) strategy. Results: In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of ‘common’ deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated. Conclusions: The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.

Tyrosine Kinase Inhibitor STI571 (Imatinib) Cooperates with Wild-Type p53 on K562 Cell Line to Enhance Its Proapoptotic Effects

In order to ascertain whether p53 has a role in chronic myeloid leukemia hematopoietic progenitor response to the innovative tyrosine kinase inhibitor STI571 (Imatinib), we overexpressed a wild type (wt) p53 construct in the K562 cell line, generated from a human blast crisis and lacking endogenous p53. Wt p53 overexpression was associated with a significant reduction of bcr-abl expression levels resulting, at least in part, from post-transcriptional events affecting the stability of p210 bcr-abl fusion protein. Moreover, we demonstrated that p53 overexpression enhances the commitment to the apoptotic death fate of K562 following its in vitro exposure to 1 µM STI571. Multiple mechanisms are involved in p53 impact on K562 survival: Most importantly, we found that a greater reduction of bcr-abl transcription by STI571 was associated with the overexpression of wt p53. Further studies are required to elucidate the mechanisms involved in the transcriptional repression of bcr-abl by STI571 and p53 and in their synergic effects on the clonal hematopoiesis of chronic myeloid leukemia.

IPO-trimethylation of histone H3-lysine9 associated with P210 BCR-ABL tyrosine kinase of chronic myeloid leukaemia.

that thrombophilia screening in IVF in the general population is not cost-effective (Fàbregues et al, 2004). It is important to recognize that thrombosis, as a complication of ovarian stimulation or OHSS, is an increasing problem. As the use of ART increases, the occurrence of related thromboembolic complications must also be investigated to determine their causes. Clearly, further research is needed to identify women at risk and to develop adequate therapeutic and prophylactic measures.