The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs.

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.

Concomitant occurrence of a primary renal NHL and of a papillary urothelial ureter cancer.

In this manuscript for the first time we describe the concomitant diagnosis of primary renal non-Hodgkin lymphoma (PRL) and of a papillary urothelial cancer in a patient with megaloblastic anemia. PRL is a rare disease, since the kidney is one of the extranodal organs usually not containing lymphoid tissue. The disease usually affects adults with an average age of 60 years and slight male preponderance. Flank pain is the most common presenting symptom and different histologies have been reported. A review of literature indicated that simultaneous diagnosis of PRL and papillary urothelial carcinoma of the urether, makes our case unique. The early diagnosis of both diseases allowed the eradication of the two neoplasms by nephro-ureterecthomy and by performing subsequent systemic chemotherapy.

Adenylate cyclase/cAMP pathway downmodulation counteracts apoptosis induced by IFN-alpha in human epidermoid cancer cells.

We have reported previously that interferon-alpha (IFN-alpha) induces apoptosis that is counteracted by an epidermal growth factor (EGF) --> Ras --> extracellular signal-regulated kinase (ERK)-dependent survival response in human epidermoid cancer KB cells. We have studied the effects of the cytokine on the cAMP-dependent pathway in these cells. A decrease in the intracellular cAMP levels was recorded in KB cells treated with IFN-alpha, whereas forskolin induced an increase in the production of cAMP that was reduced in the presence of IFN-alpha, suggesting a reduction in the activity of adenylate cyclase (AC) induced by IFN-alpha. These effects were paralleled by significant change in the expression of some AC catalytic subunit(s) and by reduction in the activity of protein kinase A (PKA). 8-Br-cAMP completely antagonized the reduction of PKA activity induced by IFN-alpha, whereas PKA inhibitor KT5720 enhanced the reduction of the enzyme activity induced by IFN-alpha. We have found that IFN-alpha induced a decrease in cAMP response element binding protein (CREB) phosphorylation without changes in its total expression. The concomitant treatment with IFN-alpha and 8-Br-cAMP potentiated and KT5720 counteracted apoptosis induced by IFN-alpha alone. In conclusion, these data suggest that the decrease in AC/cAMP pathway activity is a survival response to the apoptosis induced by IFN-alpha. Therefore, this pathway could represent a target to enhance the antitumor activity of IFN-alpha.

EGF activates an inducible survival response via the RAS-> Erk-1/2 pathway to counteract interferon-alpha-mediated apoptosis in epidermoid cancer cells.

The mechanisms of tumor cell resistance to interferon-alpha (IFNalpha) are at present mostly unsolved. We have previously demonstrated that IFNalpha induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNalpha-induced apoptosis depends upon activation of the NH(2)-terminal Jun kinase-1 (Jnk-1) and p(38) mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNalpha increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNalpha occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNalpha via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNalpha-treated cells. Moreover, IFNalpha induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNalpha. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNalpha-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNalpha-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNalpha.

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics.

The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1, 2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein-ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Spectrophotometric detection of RT-PCR-amplified BCR/ABL fusion transcripts. A survey performed on archival hematologic slides.

We describe a method of spectrophotometric detection of BCR/ABL chimeric sequences amplified by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR), enabling the use of archival hematologic slides as RNA sources. Multiplex PCR amplified b3a2, b2a2, and e1a2 break points of the BCR/ABL translocation and the normal ABL gene product. Assessment of sensitivity, performed on K562 cells, showed that the threshold approximated radioactive methods of detection (i.e., 1 positive cell in 1 x 10(6) negative cells for single round PCR and lower than 1 positive cell in 1 x 10(7) negative cells for nested PCR). Then, we assayed 38 different archival hematologic slides from 18 patients, including 11 cases of chronic myelogenous leukemia or chronic myelogenous leukemia-like disease, such as a case of myelofibrosis and a case of chronic neutrophilic leukemia, 6 cases of acute lymphoblastic leukemia, and 1 case of acute myelogenous leukemia. Amplification and spectrophotometric detection of BCR/ABL fusion messenger RNAs gave an unambiguous positive result in 24 (89%) of 27 expected positive slides, among which 17 (63%) were positive after a single PCR round. Concordant unambiguous results were obtained from 35 (92%) of 38 slides, as verified through parallel analyses of corresponding cryopreserved cells. Retrospective analysis on archival hematologic slides yielded identification of the presence or absence of the t(9;22) translocation and break point in 14 previously uncharacterized cases. The application of this method can help define the diagnosis of cases lacking other appropriate material and assist in the retrospective analysis of large patient series for which only smears are available.

Transient exposure to cytarabine increases peptide growth factor receptor expression and tumorigenicity of melanoma cells.

We have demonstrated that anticancer drugs at cytostatic concentrations enhance the expression and function of epidermal growth factor (EGF-R) and transferrin (TRF-R) receptors on human tumor cells. We hypothesized that these effects could represent a protective response of tumor cells to sublethal antiproliferative stimuli which could lead to enhanced growth potential. 72 hours exposure of human melanoma GLL-19 cells to 1,000 nM ara-C induced growth inhibition and increased the number of EGF-R, TRF-R and nerve growth factor receptor (NGF-R) on cell surface. Enhanced expression of beta 3 integrins CD49a, CD49c and CD49e, av integrin CD51, beta 3 integrin CD61, CD58/LFA3 and collagen IV and laminin was also detected in ara-C-treated GLL-19 cells. These changes at the tumor cell surface were paralleled by increased in vitro adhesion, invasive potential and clonogenic growth in soft agar and in vivo tumor formation. A more aggressive tumor cell phenotype is induced in human melanoma cells after transient exposure to cytostatic concentrations of ara-C.

Interferon alpha2 recombinant and epidermal growth factor modulate proliferation and hypusine synthesis in human epidermoid cancer KB cells.

We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

Reed-Sternberg cells of classical Hodgkin's disease react with the plasma cell-specific monoclonal antibody B-B4 and express human syndecan-1.

Although the cellular origin of Reed-Sternberg (RS) cells of classical Hodgkin's disease (HD) has been a controversial issue for many years, recent immunophenotypic and molecular studies have suggested that RS cells of a subset of classical HD cases may be related to B cells. To further define the immunophenotypic features and the differentiation stage of RS cells, a series of 56 HD samples, including both nodular lymphocyte predominance (LP) (eight cases) and classical HD (nodular sclerosis [NS], 32 cases; mixed cellularity [MC], 16 cases) with a non-T-cell phenotype, were evaluated for the immunohistochemical expression of the B-B4 antigen, a specific marker for terminally differentiated B cells. Because the cDNA of the B-B4 antigen encodes syndecan-1, a member of a family of transmembrane heparan sulfate proteoglycans thought to be involved in binding cells of the B lineage to the interstitial matrix, the B-B4 immunoreactivity was correlated with the expression of syndecan-1 in HD-derived cell lines (L428, KM-H2), as detected by both reverse transcriptase polymerase chain reaction (RT-PCR) studies and Western blotting. Our results show that B-B4 reacts with RS cells and their morphological variants of all cases of classical HD, irrespective of their antigenic phenotype (B, undetermined), albeit at a varying degree of cellular expression. Notably, a high reactivity and staining intensity for the B-B4 monoclonal antibody (MoAb) was restricted to tumor cells from NS HD. In cases of the latter subtype, B-B4 positivity was also found in sclerosis-trapped spindle cells (fibrocytes/fibroblasts). Conversely, the putative tumor cells of nodular LP HD were consistently unreactive with the B-B4 MoAb. Finally, we have demonstrated by RT-PCR, flow cytometry, and Western blotting that cultured RS cells, of B and undetermined phenotype, express syndecan-1 mRNA and produce a form of syndecan-1, recognized by the B-B4 MoAb, which is predominantly associated with glycosaminoglycans and is present at the cell surface. Our detection of the plasma cell-specific antigen B-B4 (syndecan-1) on tumor cells of classical HD further supports that RS cell progenitors may be related to germinal/postgerminal center mature B cells and suggests that expression of syndecan-1 may contribute to some of the typical biologic and histopathologic features of classical HD, with a special regard to the NS subtype.

Expression of the RET receptor tyrosine kinase and GDNFR-alpha in normal and leukemic human hematopoietic cells and stromal cells of the bone marrow microenvironment.

The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multisubunit surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFR-alpha) acts as the ligand-binding component. We have analyzed expression of RET and GDNFR-alpha in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154 primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation stages. Relatively low amounts of RET mRNA were found in early CD34+ hematopoietic progenitors, but RET transcripts appeared to increase after myelomonocytic maturation. No expression of RET was found in peripheral blood and tissue B and T lymphocytes. Analysis of human myelomonocytic cell lines was overall consistent with results obtained on purified normal cells. Accordingly, RET expression was mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0 and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies, RET transcripts were mainly detected in cells of postthymic and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression of GDNFR-alpha, which was detected only in 2 isolated primary samples and in 3 leukemia/lymphoma cell lines. However, GDNFR-alpha transcripts, in the absence of RET mRNA, were found in normal bone marrow stromal cells (BMSC), in BM fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their clonogenic growth and triggering the monocytic maturation of leukemic cells.

CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.

CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor alpha-chain, the alpha(M) (CD11b) chain of beta2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells evidenced that, in addition to circulating and tonsil B cells, a fraction of bone marrow myeloid precursors, erythroblasts, and subsets of megakaryocytes also express CD30L. Finally, we have shown that native CD30L expressed on primary leukemic cells is functionally active by triggering both mitogenic and antiproliferative signals on CD30+ target cells. As opposed to CD30L, only 10 of 181 primary tumors expressed CD30 mRNA or protein, rendering therefore unlikely a CD30-CD30L autocrine loop in human hematopoietic neoplasms. Taken together, our data indicate that CD30L is widely expressed from early to late stages of human hematopoiesis and suggest a regulatory role for this molecule in the interactions of normal and malignant hematopoietic cells with CD30+ immune effectors and/or microenvironmental accessory cells.

The role of eosinophils in the pathobiology of Hodgkin's disease.

BACKGROUND: Even though the presence of a prominent tissue eosinophilia represents a common histopathologic feature of Hodgkin's disease (HD), eosinophils have been mainly regarded as 'innocent' bystanders recruited and activated during the cellular reaction typical of HD. To evaluate the putative role of eosinophils or eosinophil-derived cytokines on tumor-cell regulation in HD, we have analyzed these cells for the functional expression of surface ligands (L) of the tumor necrosis factor (TNF) superfamily, whose specific receptors are known to transduce proliferation signals at the surface of Hodgkin (H) and Reed-Sternberg (RS) cells. MATERIALS AND METHODS: Eosinophils from peripheral blood of healthy donors and patients with HD, primary hypereosinophilic syndrome (HES), or secondary hypereosinophilia (HE), were purified by density gradient centrifugation and immunomagnetic depletion of residual granulocytes. RESULTS: By immunostaining and mRNA analysis, we were able to show that eosinophils from normal donors and patients with HD, HES, and HE express a number of receptors and ligands of the TNF superfamily, including CD40, CD40L, CD30L, CD95/Fas, CD95/FasL and 4-1BB. In addition, we provide evidence that cytokines regulating eosinophil proliferation and activation, i.e., interleukin (IL)-5, IL-3, and granulocyte-macrophage colony-stimulating factor, are able to enhance the cellular density of several TNF superfamily ligands and/or receptors at the surface of cultured eosinophils. Finally, we have shown that native CD40L and CD30L at the surface of purified eosinophils are functionally active and able to transduce proliferative signals on CD40+ and CD30+ target cells, including cultured H-RS cells. CONCLUSIONS: Our data suggest that eosinophils may act as important elements in the pathology of HD by providing cellular ligands for TNF-superfamily receptors (CD40, CD30, CD95/Fas) able to transduce proliferation and antiapoptotic signals at the surface of H-RS cells. The presence on eosinophils of receptors for TNF ligands expressed by activated T cells (i.e., OX40L, FasL, CD40L, 4-1BBL), also suggest that eosinophils may contribute to the deregulated network of interactive signals between H-RS cells, T cells, and other surrounding reactive cells.

When a module is also a domain: the rôle of the N terminus in the stability and the dynamics of immunoglobulin domains from titin.

In the course of a structural study of titin, a giant modular protein from muscle, we have reported that N-terminal extension of immunoglobulin-like (Ig-like) domains from titin stabilizes this fold. In order to investigate the structural basis of such an effect, we have solved the structure of NEXTM5, which has six amino acids added to the sequence of M5, a domain for which full structure determination has been previously achieved. In the present work, the structures and the dynamics of M5 and NEXTM5 are compared in the light of data collected for these and other titin domains. In NEXTM5, three out of the six added residues are structured and pack against the nearby BC and FG loops. As a consequence, three new backbone hydrogen bonds are formed with the B strand, extending the A strand by two residues and decreasing the exposed surface area of the loops. Additional contacts which involve the side-chains give rise to a remarkable pH dependence of the stability. Interestingly, no correlation is observed on the NMR time-scale between the overall dynamics of the extended domain and its increased stability. The most noticeable differences between the two constructs are localised around the N terminus, which becomes more rigid upon extension. Since a similar pattern of contacts is observed for other domains of the immunoglobulin I-set, our results are of general relevance for this protein family. Our work might also inspire a more rational approach to the investigation of domain boundaries and their influence on module stability.

Human eosinophils express functional CD30 ligand and stimulate proliferation of a Hodgkin's disease cell line.

The presence of a prominent tissue eosinophilia represents a typical histopathologic hallmark of Hodgkin's disease (HD). To evaluate the putative role of eosinophils on tumor cell regulation in HD, we have analyzed these cells for the functional expression of CD30 ligand (CD30L), a surface molecule able to transduce CD30-mediated proliferation signals on Hodgkin's (H) and Reed-Sternberg (RS) cells. The results demonstrate that circulating and tissue eosinophils from normal donors and patients with HD or hypereosinophilic syndrome (HES), display CD30L mRNA and express CD30L protein, as shown by immunostaining with a specific monoclonal antibody (M80) and with a biotinylated soluble CD30-Fc fusion protein. The surface density of CD30L on eosinophils from HD and HES patients was remarkably higher compared with healthy donors, probably reflecting a cytokine-mediated upregulation in these pathologic conditions. Accordingly, we provide evidence that cytokines regulating eosinophils proliferation and activation, ie, interleukin-5 (IL-5), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF), are able to enhance the cellular density of CD30L on purified eosinophils from normal subjects. Finally, we show that native CD30L on human eosinophils is a functionally active surface structure able to transduce proliferative signals on CD30+ target cells, including cultured H-RS cells. Our data suggest that eosinophils may not merely represent innocent bystanders, but rather act as important elements in the pathology of HD by contributing to the deregulated network of CD30/CD30L-mediated interactive signals between H-RS cells and surrounding reactive cells.

Immunophenotypic and molecular genetic characterization of a case of CD8+ B cell chronic lymphocytic leukemia.

We report the case of a patient with chronic B lymphocytic leukemia, with stable clinical and hematological conditions in the absence of any treatment. The flow cytometry analysis of the patient's cells revealed a CD19+, CD5+, IgM+, IgD+, lambda chain-phenotype, along with an unusual expression of CD8 alpha/alpha homodimer. B cells did not stain with the anti CD8 beta monoclonal antibody T8/2T8 5H7. Molecular biology analyses confirmed the monoclonal rearrangements of immunoglobulin heavy and lambda light chain genes in the presence of a germline configuration of T cell receptor genes. Moreover, an abnormal configuration of the CD8A gene was found in the CD8+B lymphocytic clone. We suggest that the aberrant expression of the CD8 gene could be related to its abnormal configuration.

Prognosis of chronic myelomonocytic leukemia.

BACKGROUND: Cytopenia caused by ineffective hematopoiesis and monocyte overproduction coexist in CMML, providing grounds for discussion to supporters of a dysplastic versus a proliferative identity for CMML. Follow-up information from a large series of patients may contribute to clarifying the position of this infrequent disease. METHODS: We analyzed data from 77 patients followed in five institutions. Thirty-two variables were studied for their influence on survival and on progression to acute leukemia by univariate and multivariate analysis. For some parameters, we performed a quartile analysis to reveal a possible non-monotonic influence on survival. RESULTS: Median survival was 17 months. Evolution to acute leukemia (ANLL) occurred in 11 patients (14%) within a median time of 8 months. Multivariate analysis assigned a poorer prognosis to patients presenting with thrombocytopenia, anemia and leukocytosis. Thrombocytopenia and the presence of circulating blasts were risk factors for transformation to ANLL, while raised serum aspartate transaminase at diagnosis seemed to be associated with a lower probability of blastic evolution. The Bournemouth score for CMML proved to be a valid tool for predicting survival but not acute transformation. CONCLUSIONS: CMML is a severe disease. The prognostic independence of cytopenia (anemia, thrombocytopenia) and leukocytosis underlines the coexistence of aspects typical of myelodysplastic and myeloproliferative syndromes.

Human osteoclasts and preosteoclast cells (FLG 29.1) express functional c-kit receptors and interact with osteoblast and stromal cells via membrane-bound stem cell factor.

Bone remodeling requires cooperation between osteoclasts and other specialized or accessory bone cell populations by mechanisms that have not been completely elucidated. Here we describe the expression and functional role of the proto-oncogene c-kit and of its specific ligand stem cell factor (SCF) on human osteoclasts, osteoblasts, and stromal cells derived from different sources. Our results indicate that primary osteoclasts in imprints of metaphyseal bone and giant cell tumors (GCTs) of bone, as well as a bone marrow-derived preosteoclast cell line of human origin (FLG 29.1), expressed immunodetectable c-kit protein. In contrast, tissue osteoclasts did not react with anti-SCF antibodies, and barely detectable levels of SCF mRNA and protein were found in FLG 29.1 cells. Conversely, a strong expression of membrane bound-SCF was found in primary cultured bone marrow stromal cells, in a stromal cell line (C433) derived from the mononuclear component of GCT of bone, and in a human cell line with osteoblast features (Saos-2). FLG 29.1 preosteoclast cells displayed about 29,000 binding sites/cell of a single class of high affinity c-kit receptors (Kd 6.12 x 10(-10) mol/L) with a molecular weight of about 140 kDa, along with a structurally normal c-kit mRNA. Proliferation of FLG 29.1 preosteoclast cells was stimulated by exogenous SCF, indicating that c-kit was capable of transducing growth signals. Finally, in vitro adhesion of FLG 29.1 cells to primary bone marrow stromal cells, GCT-derived stromal cells (C433), and Saos-2 osteoblast cells was significantly inhibited by an excess of soluble SCF or by monoclonal antibodies recognizing SCF binding sites on the c-kit receptor. These results indicate that c-kit is constitutively expressed on human osteoclasts and that it may be directly implicated in cell contact-dependent interaction of osteoclasts with other specialized or accessory cell populations of the bone microenvironment. Our observations suggest a role for SCF in human diseases characterized by abnormal bone resorption and remodeling.

Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity.

BACKGROUND: The giant muscle protein titin forms a filament which spans half of the sarcomere and performs, along its length, quite diverse functions. The region of titin located in the sarcomere I-band is believed to play a major role in extensibility and passive elasticity of muscle. In the I-band, the titin sequence consists mostly of repetitive motifs of tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. The highly repetitive titin architecture suggests that the molecular basis of its mechanical properties be approached through the characterization of the isolated components of the I-band and their interfaces. In the present paper, we report on the structure determination in solution of a representative Ig module from the I-band (I27) as solved by NMR techniques. RESULTS: The structure of I27 consists of a beta sandwich formed by two four-stranded sheets (named ABED and A'GFC). This fold belongs to the intermediate frame (I frame) of the immunoglobulin superfamily. Comparison of I27 with another titin module from the region located in the M-line (M5) shows that two loops (between the B and C and the F and G strands) are shorter in I27, conferring a less elongated appearance to this structure. Such a feature is specific to the Ig domains in the I-band and might therefore be related to the functions of the protein in this region. The structure of tandem Ig domains as modeled from I27 suggests the presence of hinge regions connecting contiguous modules. CONCLUSIONS: We suggest that titin Ig domains in the I-band function as extensible components of muscle elasticity by stretching the hinge regions.