Psychosocial outcome following genetic risk counselling for familial colorectal cancer. A comparison of affected patients and family members.

Few studies have reported prospective data on psychosocial outcomes after genetic counselling in families with suspected hereditary non-polyposis colorectal cancer (HNPCC). This prospective study examines the impact of multidisciplinary risk counselling on the psychosocial outcome of 139 affected cancer patients and 233 family members without cancer at risk for HNPCC. Participants completed questionnaires specific to HNPCC before and 8 weeks after attending the familial cancer clinic. Affected patients' levels of distress were closely related to their health status and exceeded that of unaffected individuals, as did worry regarding their relatives' risk. A significant reduction in general anxiety (Hospital Anxiety and Depression Scale), distress specific to familial CRC (Impact of Events Scale) and general cancer worry (Distress Hereditary Disorder) was demonstrated after counselling in both affected patients and unaffected individuals. Reduction in distress was more pronounced in affected patients given a high risk of HNPCC compared with those at intermediate risk. Among unaffected individuals, distress declined regardless of what clinical risk they were assigned. Their perceptions of risk and cancer-related threat declined, while confidence in effective surveillance increased. These results suggest the beneficial effects of multidisciplinary counselling even when high-risk information is conveyed. A patient's previous cancer experience is likely to contribute to clinically relevant distress (15% of those patients), indicating the need for appropriate counselling.

Bortezomib before and after high-dose therapy in myeloma: long-term results from the phase III HOVON-65/GMMG-HD4 trial.

The Dutch-Belgian Cooperative Trial Group for Hematology Oncology Group-65/German-speaking Myeloma Multicenter Group-HD4 (HOVON-65/GMMG-HD4) phase III trial compared bortezomib (BTZ) before and after high-dose melphalan and autologous stem cell transplantation (HDM, PAD arm) compared with classical cytotoxic agents prior and thalidomide after HDM (VAD arm) in multiple myeloma (MM) patients aged 18-65 years. Here, the long-term follow-up and data on second primary malignancies (SPM) are presented. After a median follow-up of 96 months, progression-free survival (censored at allogeneic transplantation, PFS) remained significantly prolonged in the PAD versus VAD arm (hazard ratio (HR)=0.76, 95% confidence interval (95% CI) of 0.65-0.89, P=0.001). Overall survival (OS) was similar in the PAD versus VAD arm (HR=0.89, 95% CI: 0.74-1.08, P=0.24). The incidence of SPM were similar between the two arms (7% each, P=0.73). The negative prognostic effects of the cytogenetic aberration deletion 17p13 (clone size ⩾10%) and renal impairment at baseline (serum creatinine >2 mg dl-1) on PFS and OS remained abrogated in the PAD but not VAD arm. OS from first relapse/progression was similar between the study arms (HR=1.02, P=0.85). In conclusion, the survival benefit with BTZ induction/maintenance compared with classical cytotoxic agents and thalidomide maintenance is maintained without an increased risk of SPM.

Genetic factors influencing the risk of multiple myeloma bone disease.

A major complication of multiple myeloma (MM) is the development of osteolytic lesions, fractures and bone pain. To identify genetic variants influencing the development of MM bone disease (MBD), we analyzed MM patients of European ancestry (totaling 3774), which had been radiologically surveyed for MBD. Each patient had been genotyped for ~6 00 000 single-nucleotide polymorphisms with genotypes for six million common variants imputed using 1000 Genomes Project and UK10K as reference. We identified a locus at 8q24.12 for MBD (rs4407910, OPG/TNFRSF11B, odds ratio=1.38, P=4.09 × 10(-9)) and a promising association at 19q13.43 (rs74676832, odds ratio=1.97, P=9.33 × 10(-7)). Our findings demonstrate that germline variation influences MBD and highlights the importance of RANK/RANKL/OPG pathway in MBD development. These findings will contribute to the development of future strategies for prevention of MBD in the early precancerous phases of MM.

Phase III trial of bortezomib, cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma.

We aimed at demonstrating non-inferiority of bortezomib/cyclophosphamide/dexamethasone (VCD) compared to bortezomib/doxorubicin/dexamethasone (PAd) induction therapy with respect to very good partial response rates or better (⩾VGPR) in 504 newly diagnosed, transplant-eligible multiple myeloma patients. VCD was found to be non-inferior to PAd with respect to ⩾VGPR rates (37.0 versus 34.3%, P=0.001). The rates of progressive disease (PD) were 0.4% (VCD) versus 4.8% (PAd; P=0.003). In the PAd arm, 11 of 12 patients with PD had either renal impairment (creatinine ⩾2 mg/dl) at diagnosis or the cytogenetic abnormality gain 1q21, whereas no PD was observed in these subgroups in the VCD arm. Leukocytopenia/neutropenia (⩾3°) occurred more frequently in the VCD arm (35.2% versus 11.3%, P<0.001). Neuropathy rates (⩾2°) were higher in the PAd group (14.9 versus 8.4%, P=0.03). Serious adverse events, both overall and those related to thromboembolic events, were higher in the PAd group (32.7 versus 24.0%, P=0.04 and 2.8 versus 0.4%, P=0.04). Stem cell collection was not impeded by VCD. VCD is as effective as PAd in terms of achieving ⩾VGPR rates with fewer PD and has a favorable toxicity profile. Therefore, VCD is preferable to PAd as induction therapy.

A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates.

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes. Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

A plant homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor.

We have isolated cDNA clones of Spinacea oleracea L. and Oenothera hookeri of 930 and 1017 base pairs, respectively. The open reading frame deduced from the Oenothera sequence codes for a protein of a calculated molecular mass of 29,200. The primary amino acid sequence exhibits a very high degree (88%) of homology to the 14-3-3 protein from bovine brain, and protein kinase C inhibitor from sheep brain. Subsequently the plant protein was partially purified from leaf extract. The partially purified plant protein inhibited protein kinase C from sheep brain in a heterologous assay system. The active fraction consisted of 5-6 different polypeptides of similar molecular size. One of these proteins crossreacted with a peptide-specific antibody against protein kinase C inhibitor protein from sheep brain.

Compartmentalization of TNF-related apoptosis-inducing ligand (TRAIL) death receptor functions: emerging role of nuclear TRAIL-R2.

Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.

Modulation of CD4+ T-cell activation by CD95 co-stimulation.

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

Bone morphogenic protein 6: a member of a novel class of prognostic factors expressed by normal and malignant plasma cells inhibiting proliferation and angiogenesis.

Pathogenesis of multiple myeloma is associated with an aberrant expression of pro-proliferative, pro-angiogenic and bone-metabolism-modifying factors by malignant plasma cells. Given the frequently long time span from diagnosis of early-stage plasma cell dyscrasias to overt myeloma and the mostly low proliferation rate of malignant plasma cells, we hypothesize these to similarly express a novel class of inhibitory factors of potential prognostic relevance. Bone morphogenic proteins (BMPs) represent possible candidates as they inhibit proliferation, stimulate bone formation and have an effect on the survival of cancer patients. We assessed the expression of BMPs and their receptors by Affymetrix DNA microarrays (n=779) including CD138-purified primary myeloma cell samples (n=635) of previously untreated patients. BMP6 is the only BMP expressed by malignant and normal plasma cells. Its expression is significantly lower in proliferating myeloma cells, myeloma cell lines or plasmablasts. BMP6 significantly inhibits the proliferation of myeloma cell lines, survival of primary myeloma cells and in vitro angiogenesis. A high BMP6 expression in primary myeloma cell samples delineates significantly superior overall survival for patients undergoing high-dose chemotherapy independent of conventional prognostic factors (International Staging System (ISS) stage, beta(2) microglobulin).

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis.

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains. To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble lambda-phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplas cpn10 proteins in two cases restored lambda-phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn10 showed significantly reduced complementation efficiencies.

Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants.

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.

Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site.

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.