Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling.

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.

Particle size fractionation of paralytic shellfish toxins (PSTs): seasonal distribution and bacterial production in the St Lawrence estuary, Canada.

We determined the seasonal distribution of paralytic shellfish toxins (PSTs) and PST producing bacteria in > 15, 5-15, and 0.22-5 microm size fractions in the St Lawrence. We also measured PSTs in a local population of Mytilus edulis. PST concentrations were determined in each size fraction and in laboratory incubations of sub-samples by high performance liquid chromatography (HPLC), including the rigorous elimination of suspected toxin 'imposter' peaks. Mussel toxin levels were determined by mouse bioassay and HPLC. PSTs were detected in all size fractions during the summer sampling season, with 47% of the water column toxin levels associated with particles smaller than Alexandrium tamarense (< 15 microm). Even in the > 15 microm size fraction, we estimated that as much as 92% of PSTs could be associated with particles other than A. tamarense. Our results stress the importance of taking into account the potential presence of PSTs in size fractions other than that containing the known algal producer when attempting to model shellfish intoxication, especially during years of low cell abundance. Finally, our HPLC results confirmed the presence of bacteria capable of autonomous PST production in the St Lawrence as well as demonstrating their regular presence and apparent diversity in the plankton.

Characterization of mediator complexes from HeLa cell nuclear extract.

A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (approximately 500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (approximately 2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at approximately 500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 x 10(5) to 6 x 10(5) molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the approximately 2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The approximately 2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

Quality assurance of progenitor cell content of apheresis products: a comparison of clonogenic assays and CD34+ enumeration. The Canadian Apheresis Group and Canadian Bone Marrow.

The most common methods used for evaluation of the haematopoietic stem cell content of peripheral blood apheresis products are the colony forming cell assay and the enumeration of CD34+ cells by flow cytometry. The Canadian Apheresis Group and the Canadian Bone Marrow Transplant Group established a multicentre study to compare the reproducibility of colony forming cell assays and CD34+ enumeration by flow cytometry in six transplant centres routinely performing haematopoietic stem cell apheresis. Over a 5-month period in 1996, 31 fresh apheresis samples were shipped by overnight courier for testing at six centres to perform CD34+ enumeration by flow cytometry and clonogenic assays. The mean coefficient of variation and range for the following assays were: cell count 36% (2.6-148%), CFU-GM 82% (46-123%), CD34+ absolute/kg 60% (14-174%) and CD34+ per cent 42% (12-84%). The wide variation in cell count in this pilot study highlights the difficulties related to provision of samples for quality assessment programmes. Results showed poor interinstitutional reproducibility even among selected samples with similar cell counts for both CFC and CD34+ assays demonstrating the need for development and implementation of an interinstitutional quality assurance programme for haematopoietic stem cell assessment. Provision of a reliable source of testing material will be a necessary next step.

A prospective study of G-CSF effects on hemostasis in allogeneic blood stem cell donors.

Granulocyte colony-stimulating factor (G-CSF) is used in healthy donors of peripheral blood stem cells (PBSC) for allogeneic transplantation. However, some data have recently suggested that G-CSF may induce a hypercoagulable state, prompting us to study prospectively 22 PBSC donors before and after G-CSF 5 microg/kg twice daily. We sought evidence for changes in the following parameters: platelet count, von Willebrand factor antigen (vWF:Ag) and activity (vWF activity), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), platelet activation markers (GMP-140 and PAC-1), activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulant factor VIII (FVIII:C), thrombin-antithrombin complex (TAT), prothrombin fragment 1+2 (F1+2), thrombomodulin (TM) and tissue plasminogen activator antigen (tPA:Ag) prior to G-CSF and immediately before leukapheresis. ADP-induced platelet aggregation studies were also performed. G-CSF administration produced only mild discomfort. We found a significant increase in vWF:Ag (from 0.99 +/- 0.32 U/ml to 1.83 +/- 0.69 U/ml; P < 0.001), in vWF activity (from 1.04 +/- 0.34 U/ml to 1.78 +/- 0.50 U/ml; P < 0.001) and in FVIII:C (from 1.12 +/- 0.37 U/ml to 1.73 +/- 0.57 U/ml; P < 0.001) after G-CSF. Of note, four donors with low baseline vWF had a two- to three-fold increase after receiving G-CSF. G-CSF had no impact on the platelet count, beta-TG, PF-4, GMP-140 or PAC-1. The final% of platelet aggregation decreased from 73 +/- 22% to 37 +/- 26% after G-CSF (P < 0.001). We found a significant decrease in aPTT after G-CSF (29.9 +/- 3.1 s to 28.3 +/- 3.3 s; P = 0.004), but the PT was unaffected. In addition, we also observed a significant increase in TAT, F1+2 and TM, but not in tPA:Ag. Our data suggest that G-CSF may possibly induce a hypercoagulable state by increasing levels of FVIII:C and thrombin generation. In contrast to this information, we found reduced platelet aggregation after G-CSF administration. The clinical implications of these findings remain unclear and larger studies are definitely required.

Two different schedules for integrating filgrastim as adjuvant therapy in the treatment of patients with advanced stage Hodgkin's lymphoma receiving MOPP/ABV hybrid chemotherapy.

PURPOSE: Management of advanced-stage Hodgkin's disease with a MOPP/ABV hybrid regimen (mechlorethamine, vincristine, procarbazine, prednisone, Adriamycin, bleomycin and vinblastine) has yielded a high complete response rate (75-85%). However, myelosuppression can limit delivery of treatment. Filgrastim has been shown to reduce chemotherapy-related neutropenia and allow for on-time administration of planned doses of chemotherapeutic agents. The objective of this study was to find the best way to integrate filgrastim with the MOPP/ABV hybrid regimen. METHODS: Enrolled in this study were 24 patients (aged 18-52 years) with newly diagnosed, histologically documented Hodgkin's disease. In schedule I, patients received filgrastim (5 microg/kg s.c. daily) beginning on day 9, 24 h after administration of ABV. In schedule II, patients received filgrastim concomitantly with procarbazine on days 2-7 (starting 24 h after day-1 MOPP administration and stopping 24 h before ABV administration) as well as after ABV beginning on day 9. Filgrastim after ABV administration was administered until two consecutive ANC readings of 10 x 10(9)/l were achieved. RESULTS: All patients were able to complete all six cycles of therapy. There was a trend to fewer dose reductions in schedule II (0.76%) as compared to schedule I (4.2%) with a P-value of 0.077 (chi-squared test). Specifically, 11.6% of MOPP courses and 5.5% of ABV courses were dose-reduced in schedule I versus 1.7% and 1.4%, respectively, in schedule II. CONCLUSION: In conclusion, filgrastim was effective in supporting the delivery of the MOPP/ABV chemotherapy. Concomitant administration of filgrastim with procarbazine (days 2-7) appears to be safe and allows the maximum dose intensity of this therapy.

A genome-wide functional assay of signal transduction in living mammalian cells.

We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli. A promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones. When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes. Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation. Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes. Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest.

Treatment of essential thrombocythemia during pregnancy with interferon-alpha.

BACKGROUND: Only a few cases of essential thrombocythemia in pregnant women have been reported, and the management of this myeloproliferative disorder during pregnancy remains uncertain. We report a successful pregnancy in a patient who had essential thrombocythemia and who was treated with interferon-alpha, and we review the literature for the outcome of similar patients. CASE: A 32-year-old woman, gravida 4, para 3, aborta 0, presented at 18 weeks' gestation with two episodes of amaurosis fugax and an elevated platelet count of 2300 x 10(9)/L. The initiation of interferon-alpha led to a progressive fall of the platelet level, with no occurrence of thrombotic or hemorrhagic manifestations. Serial ultrasound examinations revealed normal fetal and placental development. The patient was delivered of a male infant at 37 weeks. Both child and placenta were normal on examination. CONCLUSION: Our case and the current available data suggest that interferon-alpha may be the best therapeutic option for pregnant patients with essential thrombocythemia in whom myelosuppression is required.

Granulocyte recovery after sequential transfusion of mobilized blood stem cell concentrates in syngeneic recipients.

Three patients received sequential transfusions of G-CSF-mobilized peripheral blood stem cells from their identical twin in an attempt to abrogate neutropenia. Blood stem cells were harvested by leukapheresis in the healthy donor twins following mobilization with rhG-CSF at 5 micrograms/kg/day subcutaneously for at least 5 days. An average of 2.2 x 10(7) CFU-GM (range: 1.4-3.3) were collected and transfused without further manipulation. One patient, transfused with a CFU-GM dose of 3 x 10(7) on day +6 after a syngeneic marrow transplant, experienced near-complete elimination of absolute neutropenia until spontaneous engraftment occurred on day +11. In the other two patients, we unexpectedly observed a transient granulopoietic inhibition, possibly related to the high T cell content of the blood stem cell transfusions.

Hematopoietic engraftment from a minimal number of apheresis procedures after mobilization of peripheral blood stem cells with chemotherapy and rhG-CSF.

In a cohort of 13 patients, peripheral blood stem cells (PBSC) were harvested by apheresis after mobilization with chemotherapy and rhG-CSF. Nine patients who had excellent mobilization were transplanted with PBSC concentrates from a minimal number of apheresis procedures (mean of 1.5, range = 1-3). During collection, the number of circulating progenitors was on average 50 times higher than those observed at the steady state in the peripheral blood of healthy unstimulated individuals. The mean number of CFU-GM/kg reinfused per patient was 28.1 x 10(4) (range = 18.0-50 x 10(4)). The use of rhG-CSF, at either 1 or 5 micrograms/kg/day, resulted in a significantly greater yield of CFU-GM per mononuclear cells than that observed previously in a comparable group of patients receiving chemotherapy alone. Prompt and durable engraftment occurred after myeloablative chemotherapy. The average duration of absolute neutropenia was 9 days. Transfusion requirements were low with an average of four packed red cell units and two platelet transfusions per patient. The shortest follow-up is 5 months and the longest is 20+ months. The convenience of this new approach to support myeloablative therapy offers new possibilities for the administration of a higher dose-intensity of chemotherapeutic agents. A limited number of apheresis procedures timely harvested will improve the cost effectiveness of transplant programs.

Blood-derived stem cell collection in acute nonlymphoblastic leukemia: predictive factors for a good yield.

Blood-derived stem cell (BDSC) autografting represents an interesting theoretical approach to the treatment of acute nonlymphoblastic leukemia (ANLL). The feasibility and safety of this procedure have now been established by several observations of rapid hematopoietic recovery following high-dose chemotherapy and autologous reinfusion of BDSCs harvested during remission. Using clonogenic assays for granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte erythrocyte monocyte colony-forming units (CFU-GEM), we tested peripheral blood samples from 20 consecutive patients recovering from induction chemotherapy for ANLL. Results were correlated with other clinical and hematological factors in order to define optimal criteria for successful BDSC harvesting. Two different patterns of increment were observed in the number of peripheral blood progenitor cells during early recovery from chemotherapy in our patients, suggesting that a total period of 10-12 days should be covered for ideal BDSC harvesting by cytapheresis. Associated clinical factors that appear predictive for a better yield are: 1) chemotherapy with daunorubicin, cytosine arabinoside, and thioguanine (DAT), 2) synchronous recovery of monocytes and platelets, and 3) a good overall performance status of the patient. Complete remission status and absence of ongoing infection should also be considered when selecting patients for BDSC harvesting. No correlation was found between the number of circulating CFUs, as indicated by the clonogenic assays, and that of either My-10- or HLA-Dr-positive cells identified by immunofluorescence on the same blood samples. In this study, less than 50% of patients with newly diagnosed ANLL would have been considered good candidates for a BDSC harvesting program.

Microgranular acute promyelocytic leukemia: a proposed role for a greater deformability of the leukemic cell.

A case of microgranular acute promyelocytic leukemia (APL), M-3 variant, is reported in a boy aged 5 years. The disease, which was rapidly fatal, presented with acute disseminated intravascular coagulation (DIC) and leukocytosis. Different cytomorphologic subtypes of promyelocytes were identified on the basis of cytoplasmic granular patterns: the microgranular type with barely visible cytoplasmic granulations and deeply basophilic cytoplasm and the more characteristic type with large promyelocytes containing azurophil granules. We observed a ratio of large promyelocytes to microgranular promyelocytes of 1:1.2 in the marrow and 1:4 in the peripheral blood. To explain this discrepancy, we hypothesize that the microgranular promyelocytes may be more deformable than the typical promyelocyte and that this intrinsic cellular characteristic may promote marrow egress and increase the likelihood of hyperleukocytosis in the M-3 variant.

High-dose cytosine arabinoside for acute nonlymphocytic leukemia.

Eighteen patients with acute nonlymphocytic leukemia (ANLL), aged 17-73 years, were treated with high-dose cytosine arabinoside (HD-Ara-C) using 3 g/m2 IV q 12 hours X 12 doses. Seven patients were treated for relapse and four (57%) obtained a complete remission with a median duration of 19.5 weeks. In nine patients, refractory to conventional chemotherapy, no complete responders were observed. Treatment failure was most commonly due to drug resistance. Two elderly patients with ANLL not previously exposed to chemotherapy died during the initial induction. Recent data on the HD-Ara-C experience in ANLL are presented and compared with this study.

Anti-PP1Pk and early abortion.

Anti-PP1Pk is found in the serum of individuals of phenotype p. Recurrent first trimester miscarriages have been reported in nearly 50 percent of mothers with the p phenotype. A comparative serologic study of three p phenotype mothers is reported. The proposita had three miscarriages while her two identical twin sisters had normal pregnancies. Following serum fractionation on DEAE-cellulose, serial agglutination with P1 and P2 erythrocytes indicated a possible association between IgG anti-P and the increased risk of abortion.