Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to nutlin-3 induces a characteristic gene expression profile, which correlates with nutlin-3-mediated cytotoxicity.

By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.

Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin.

Although several studies have shown that the serum levels of osteoprotegerin (OPG) are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC). In addition, bone marrow mesenchymal stem cell (MSC)-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSC-derived endothelial cells (EC) or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.

Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin.

Although several studies have shown that the serum levels of osteoprotegerin (OPG) are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC). In addition, bone marrow mesenchymal stem cell (MSC)-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSC-derived endothelial cells (EC) or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.

Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function.

OBJECTIVE: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. PATIENTS: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. RESULTS: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C-independent activities (24-38%) when compared with wild-type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 +/- 30.8 nM, rPSwt; Kd 15.2 +/- 0.9 nM) as well as to Ca2+-dependent conformation-specific monoclonal antibodies for GLA domain was significantly reduced. CONCLUSIONS: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.

Biophenotypes and survival of BRCA1 and TP53 deleted breast cancer in young women.

The aim of this study was to examine the loss of heterozygosity (LOH) of BRCA1 (17q21) and TP53 (17p13.1) in early-onset breast cancer patients; to correlate biopathological characteristics with molecular alterations; and to investigate the survival of LOH-related cancers. BRCA1 and TP53 LOH were evaluated in 78 early-onset breast cancers (< or = 40 years, Group 1) and 80 patients with age > 55 years (Group 2). Cases were characterized for multiple biological markers (ER, PR, proliferation index (PI), NEU and p53). LOH was carried out on microdissected paraffin embedded tissues; microsatellites D17S855 (BRCA1) and D17S786 (TP53) were amplified by fluorescent PCR and analyzed by an automated DNA sequencer. Early-onset breast cancers showed a higher frequency of ductal histotype (89.7% vs. 56.3% p < 0.001), node-positive (53.8% vs. 38.7%), larger size (p = 0.017), higher mitotic rate (p = 0.025), higher nuclear and final grade (p = 0.01 and p = 0.001, respectively). D17S855 LOH was 32.8% in group 1 vs. 21% in group 2; D17S786 LOH was 50.7% vs. 31.3% (p = 0.03), respectively. BRCA1 LOH was correlated with higher PI (p = 0.032) and higher p53 expression (p < 0.001) in group 1 and with higher NEU expression (p = 0.028) in group 2. TP53 LOH was correlated with p53 overexpression (p = 0.03) in group 1. A worse clinical outcome in early-onset LOH related cancers emerged from follow-up data: TP53 and BRCA1 LOH were associated with a shorter relapse free interval (RFI) (p = 0.03) and a poorer overall survival (OS) (p = 0.04), respectively. This study underlines different biological profiles in the two age groups investigated, probably reflecting different mechanisms of carcinogenesis. In accordance with adverse histopathological features in early-onset patients, LOH-related cancers have an unfavorable prognosis.

Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms.

The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the beta isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both alpha (PPP2R1A) and beta isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.

Refinement of the LOH region 1 at 11q23.1 deleted in human breast carcinomas and sublocalization of 11 expressed sequence tags within the refined region.

Loss of constitutive heterozygosity at 11q23 has been detected in various human solid tumors. Here, we described the analysis of a series of normal and tumor pairs from 110 breast carcinomas for the presence of loss of heterozygosity at 11q23 loci. The overall frequency of LOH was 48%, confirming the importance of deletions at 11q23 in breast tumorigenesis. Previously, we have identified two independent regions of LOH at 11q23, the LOH region 1 at 11q23.1 and the LOH region 2 at 11q23.3. The most telomeric region was recently refined between loci D11S1345 and D11S1316, a region of about 1 Mb. However, the LOH region 1, most centromeric, was still not finely refined: the boundaries were defined by loci D11S2000 and D11S897, separated by about 8 Mb. Here, we refined its boundaries between loci D11S1347 and D11S927, a region of about 2 Mb. We have mapped 11 expressed sequence tags (ESTs) within this region and excluded another 20. This study represents a further step toward the identification of the putative tumor suppressor gene found within the LOH region 1 at 11q23.1.

Risk of venous thromboembolism and stroke associated with oral contraceptives. Role of congenital thrombophilias.

To assess the risk of thromboembolism in women using oral contraceptives (OCs), we identified through computer search in the hospitals of the province of Parma, Italy, all women aged 15-44 who were resident in the province and had a documented thromboembolic event in the years 1989-93. The number of users and nonusers of OCs was estimated by the drug sale data for the province and by the demographic statistics. In cases with venous thromboembolism (VT) the prevalence of concomitant deficiency of antithrombin III, protein C, protein S, and of factor V gene mutation Arg506GIn was evaluated. The incidence rate of VT was 37/59,603 woman-years in users (0.62 per 1000) and 13/303,954 woman-years in nonusers (0.042 per 1000), for a relative risk (RR) of 14.5 (95% confidence interval: 7.8-27.1; P < 0.001); the rate of stroke per 1000 woman-years was 0.17 in users and 0.036 in nonusers (RR = 4.6; 2.9-10.7; P < 0.01). A congenital thrombophilia involving the protein C anticoagulant system was documented in about 25% of young women developing venous thromboembolism while on OCs.

Mutation pattern in clinically asymptomatic coagulation factor VII deficiency.

A total of 122 subjects, referred after presurgery screening or checkup for prolonged prothrombin time, were characterized for the presence of coagulation factor VII deficiency. Fourteen subjects carried a partial and asymptomatic deficiency, and in half of them dysfunctional molecules were detected in plasma. In nine subjects we found five missense mutations differing from those previously found in factor VII deficient patients. The others were homozygous for a common polymorphism (R353Q) that affects factor VII levels. A new codon dimorphism (A330) was also found in exon 8. Four mutations (R223W, M298I, R304Q, and R353Q) located at FVII-specific residues point out protein regions that are important for coagulation factor evolution, and two mutations (G342E and E265K) affect generic or partially generic residues. The newly reported mutations were combined with those we previously found, totalling 17 independent mutations responsible for FVII deficiency in 27 Italian pedigrees. We observed several similarities with the mutation pattern determined in factor IX, which include a high percentage of transitions at CpG doublets, the presence of hot spot sites affected by multiple substitutions, and of several topologically equivalent mutations.

Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the second EGF-like domain.

The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glu102Lys mutation affects a residue of the second EGF-like module also conserved in protein C. Both mutated residues are surface-exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.