Glycosylated or non-glycosylated G-CSF differently influence human granulocyte functions through RhoA

Granulocyte function may be altered after in vivo G-CSF administration and this has been related to both an immaturity of mobilized cells and to a defect in F-actin polymerization. In this paper we show that in resting Filgrastim (non-glycosylated G-CSF)-pulsed cells, F-actin polymerization, membrane-linked RhoA and cell polarization are enhanced compared to those found in resting Lenograstim (glycosylated G-CSF)-cells. The basal hyper-activation of RhoA could be responsible for the morphological and functional modifications of Filgrastim-mobilized cells. Moreover, Filgrastim-mobilized cells, but not Lenograstim-mobilized cells, are unable to correctly respond to LPS stimulation, as demonstrated by minor further RhoA activation and cell elongation.     

Role of carbohydrate moiety in granulocyte colony stimulating factor

Biological activities of two granulocyte-colony stimulating factor (G-CSF) preparations with (Lenograstim) or without (Filgrastim) sugar moiety were compared. Both G-CSF preparations similarly enhanced the N-Formyl-Met-Leu-Phe-induced-migration of human peripheral blood polymorphonuclear cells, but did not significantly affect the proliferation of human oral tumor cell lines (HSC-2, HSG). However, Lenograstim induced cytotoxicity (accompanied by the production of cytoplasmic vacuoles and large DNA fragments) in human promyelocytic leukemic cells HL-60, more potently than Filgrastim. Lenograstim, but not Filgrastim, enhanced the cytotoxic activity of sodium ascorbate. In contrast to Lenograstim, Filgrastim was degraded gradually, but too slowly to explain its lower biological activity. These data suggest that the carbohydrate moiety in G-CSF might confer unique biological activities.     

Inhibition of lysophosphatidic acid-induced RhoA activation and tumor cell invasion by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors

The mevalonate metabolic pathway is necessary for the isoprenylation of a number of small GTPases. We have previously presented that Rho plays a pivotal role in 1-oleoyl-lysophosphatidic acid (LPA)-induced invasion of rat ascites hepatoma MM1 cells. Herein we report the effect of HMG-CoA reductase inhibitors, fluvastatin and lovastatin, on the in vitro invasion of MM1 cells. Fluvastatin and lovastatin inhibited LPA-induced MM1 cell invasion in a dose-dependent manner. Fluvastatin inhibited LPA-induced translocation of RhoA protein from the cytosol to the membrane and RhoA activation which was measured by pull-down assay for GTP-bound RhoA. Fluvastatin also inhibited the translocation of both endogenous and dominant-active RhoA from the cytosol to the membrane, actin stress fiber assembly and in vitro invasion of the cells expressing dominant-active RhoA (Val14-RhoA). These results indicate that HMG-CoA reductase inhibitors have the potential to reduce RhoA activation and cancer cell invasion by targeting the Rho protein isoprenylation.     

Massive extramedullary disease progression in a patient with stable multiple myeloma during G-CSF priming for peripheral blood progenitor mobilization.

High dose therapy followed by autologous peripheral blood progenitor cell (PBPC) transplantation has recently become an encouraging treatment option for younger patients with multiple myeloma (MM). The influence of the growth factors used for progenitor mobilization on myeloma cells is not known. We report on a patient suffering from IgG kappa myeloma who had been in stable, very good partial remission for seven months after standard therapy until PBPC mobilization with granulocyte-colony stimulating factor (G-CSF, Filgrastim) was initiated. Massive extramedullary disease progression occured coincidentally with the administration of G-CSF. The case suggest the possibility of myeloma stimulation by G-CSF during PBPC mobilization.     

Normal neutrophil maturation is associated with selective loss of MAP kinase activation by G-CSF

Although both GM-CSF and G-CSF activate p42/44 MAPK in neutrophil progenitors, the ability of G-CSF to cause MAPK activation is lost in mature neutrophils, while GM-CSF exposure still causes activation. The mechanism of this differential effect related to maturation status has not been explored. We verified that G-CSF and GM-CSF receptors remain functional on purified mature neutrophils by demonstrating that both cytokines caused phosphorylation of STAT3. However, only GM-CSF was capable of activating MAPK as assessed by gel shift and in vitro kinase assay. Both G-CSF and GM-CSF caused activation of p21 ras in neutrophils, demonstrating that early events in the ras-MAPK pathway remain functional after stimulation by either cytokine. Inhibition of tyrosine phosphatase activity by pervanadate restored the ability of G-CSF to activate MAPK in mature neutrophils. Specific inhibition of the SHP-1 phosphatase, known to be activated by G-CSF but not GM-CSF also restored the ability of G-CSF to activate MAPK in neutrophils. These studies suggest that G-CSF activation of SHP-1 may be an important regulatory step for permitting optimal terminal differentiation during neutrophil production and add to our knowledge of the instructional role of G-CSF and GM-CSF for balancing proliferation and differentiation of neutrophil progenitor cells. This information may prove useful for the understanding of conditions in which neutrophil proliferative/differentiative balancing is dysregulated, such as myeloid leukemia and myelodysplastic disorders.     

RACK1 promotes breast carcinoma migration/metastasis via activation of the RhoA/Rho kinase pathway

We aimed to gain a mechanistic understanding of the role of RACK1 in breast carcinoma migration/metastasis. Migration assays were conducted in breast carcinoma cell lines. siRNA targeting RACK1 as well as the Rho kinase inhibitor were also applied. Immunoprecipitation and immunofluorescence were used to study the RACK1/RhoA interaction. GTP-Rho pull-down assays were performed to assess the activation of RhoA. We also conducted immunohistochemistry in 160 breast carcinoma samples. Experiments in vitro showed that RACK1 promotes migration via interaction with RhoA and activation of the RhoA/Rho kinase pathway. Immunohistochemistry in 160 samples revealed that RACK1 is strongly correlated with accepted tumor spread indicators and RhoA (all P < 0.05). Kaplan–Meier survival analysis indicated a correlation between higher RACK1 expression and shorter survival times (P < 0.001). RACK1 is a prognostic factor that promotes breast carcinoma migration/metastasis by interacting with RhoA and activating the RhoA/Rho kinase pathway.     

Kinetics of neutrophil production in normal and neutropenic animals during the response to filgrastim (r-metHu G-CSF) or filgrastim SD/01 (PEG-r-metHu G-CSF).

Filgrastim G-CSF has a short, biologically active half-life, and its effective use depends on repeated inoculations. A major aim, therefore, has been to develop a once-per-chemotherapy cycle formulation. To this end, a polyethylene glycolylated form of Filgrastim, known as SD/01, has been developed. In this study, we compared the cellular kinetics of granulocyte production in mice stimulated with SD/01 and granulocyte colony-stimulating factor (G-CSF). Mice were injected with a single dose of SD/01 (1 mg/kg) or G-CSF (125 microg/kg) twice per day for 4 days. Mice rendered leukopenic with a single injection of cyclophosphamide (200 mg/kg) and temozolomide (90 mg/kg) were similarly treated at their 3-day neutrophil nadir. Tritiated thymidine was injected for autoradiographic labeling studies. Bone marrow labeling indices and the release of labeled neutrophils and monocytes into the peripheral blood were assessed. Granulocytopoiesis was stimulated similarly by both SD/01 and G-CSF in both normal and neutropenic animals, with counts rising to >20 x 10(9) polymorphonuclear neutrophils/l in both cases. Bone marrow thymidine labeling indices were increased, indicating a greater proportion of cells in DNA synthesis and an elevated proliferative activity. Compared with the normally slow release of neutrophils into the peripheral blood, labeled neutrophils (and monocytes) were rapidly released, increasing to peak levels at approximately 24 h. The peripheral half-life of neutrophils was not significantly different from normal, and the mitotic amplification factors for increase in granulocytopoiesis, accounted for by 3-3.9 extra cell divisions, were comparable for both factors. We conclude that neutrophil kinetics are stimulated in the same way and to the same extent by both SD/01 and G-CSF.     

AKAPs competing peptide HT31 disrupts the inhibitory effect of PKA on RhoA activity

A-kinase can inhibit RhoA activation through phosphorylating ser188 of RhoA. AKAP is a novel protein that can target PKA to different subcellular compartment. Evidence has been presented that PKA anchorage by AKAP is important for the kinase to exert its function. This study analyzed the role of PKA anchorage in PKA-induced antagonism against RhoA activity and function. The cells transfected with pcDNA HT31wt/mut were treated with LPA and/or CPT-cAMP. The amount of GTP-RhoA and phosphorylation of RhoA was detected by Western blotting with specific antibodies. The formation of stress fiber was visualized under fluorescent microscope. The gene expression activity was analyzed by luciferase reporter gene assay. The motility and the anchorage-independent growth assays were carried out with stably transfected cells expressing the AKAP inhibitory peptide HT31. The results showed that HT31 not only blocked the PKA-induced phosphorylation of RhoA but also prevented the PKA-induced inhibition on RhoA activation. The disruption of PKA anchorage abolished its inhibition on the LPA-induced expression of reporter gene SRE-luciferase. The ability of PKA to antagonize the LPA-induced stress fiber formation was partly impaired upon the disruption of the PKA anchorage. The control of PKA on migration and the proliferation excited by LPA disappeared in stably transfected cells highly expressing HT31. The results revealed that PKA anchorage was necessary for the kinase to exert its inhibitory effect on RhoA activation and RhoA-dependent biological activities.     

XM02 is superior to placebo and equivalent to Neupogen™ in reducing the duration of severe neutropenia and the incidence of febrile neutropenia in cycle 1 in breast cancer patients receiving docetaxel/doxorubicin chemotherapy

Background  

Recombinant granulocyte colony-stimulating factors (G-CSFs) such as Filgrastim are used to treat chemotherapy-induced neutropenia. We investigated a new G-CSF, XM02, and compared it to Neupogen™ after myelotoxic chemotherapy in breast cancer (BC) patients.     

von Hippel-Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is associated with the development of vascular tumors including renal cell carcinoma. Aside from the role played by the VHL protein (pVHL) in negative regulation of hypoxia-inducible factor, 41F-1alpha, pVHL also takes part in cytoskeletal organization. Thrombin is a serine protease involved in angiogenesis and in cancer progression and its action is mediated by the protease-activated receptors (PARs). In several cell types, thrombin induces reorganization of the cytoskeleton along with RhoA activation. Thus, we conducted an investigation on the capacity of thrombin to regulate pVHL expression. Our results demonstrated that VHL mRNA and protein levels were increased by thrombin in cultured renal cancer cells. Cytoplasmic pVHL was redistributed to perinuclear regions and membrane fractions following thrombin treatments. Stimulation of Caki-1 cells with PAR1, PAR2 and PAR4 agonist peptides demonstrated that PAR1 was the receptor involved in thrombin-induced pVHL expression. Western blot analysis confirmed that these cells express PAR1 and that its expression was increased by thrombin. PAR1 activation by both thrombin and an agonist peptide stimulated renal cancer cell invasion through Matrigel. Interestingly, the upregulation of pVHL was dependent on RhoA because C3 exotoxin abolished pVHL induction. However, the pharmacological Rho kinase inhibitor, Y27632, did not influence pVHL expression in the presence of thrombin, suggesting that other RhoA effectors were involved in the process. Together, these results demonstrate that thrombin induces both pVHL expression via PAR1/RhoA activation as well as the stimulation of renal cancer cell invasion suggesting a role for thrombin in tumor invasion.     

Lysophosphatidic acid stimulates PC-3 prostate cancer cell Matrigel invasion through activation of RhoA and NF-kappaB activity

This study was performed to determine the relationship of lysophosphatidic acid (LPA) stimulation and increased Ras homolog A (RhoA) activity to nuclear factor kappa B (NF-kappaB) activity, and the role of these factors in regulating prostate cancer cell invasion. PC-3 high invasive cells demonstrated constitutively increased RhoA, NF-kappaB, and in vitro Matrigel invasion which were further induced by LPA stimulation or transfection with constitutively active RhoA Q63E mutant. LPA treatment rapidly and transiently induced RhoA activity followed by maximally increased DNA binding of NF-kappaB at 1 h and AP-1 at 4 h. The LPA-induced NF-kappaB DNA binding was preceded by transient IkappaBalpha phosphorylation, and decreased total IkappaBalpha levels. Further demonstrating the relationship between RhoA and NF-kappaB activation, PC-3 cells stably transfected with constitutively active RhoA Q63E demonstrated constitutively increased phospho-IkappaBalpha, while PC-3 cells transfected with dominant negative RhoA N19 exhibited decreased phospho-IkappaBalpha levels. The LPA-induced Matrigel invasion and NF-kappaB DNA binding activity were both inhibited by expression of the RhoA inhibitor C3 exoenzyme or dominant negative mutant NF-kappaB inhibitor IkappaBalpha S32/36A. Similarly, transfection with dominant negative IkappaBalpha S32/36A inhibited PC-3 RhoA Q63E cell in vitro invasion. Treatment of PC-3 high invasive and RhoA Q63E cells with sodium salicylate or lactacystin inhibited NF-kappaB and invasion, while pyrrolidine dithiocarbamate (PDTC) treatment of PC-3 high invasive cells inhibited NF-kappaB only. Each inhibitor blocked LPA-induced invasion while PDTC inhibited LPA-induced NF-kappaB and invasion to the greatest extent. These results point to a model where LPA stimulates RhoA and increased PC-3 prostate cancer cell invasion activity through an NF-kappaB-dependent pathway.     

Differences in in vitro proliferative responsiveness to granulocyte colony-stimulating factor and interleukin 2 of bone marrow cells from mice treated with chemotherapeutic drugs.

The toxicity effects of several anticancer drugs on normal mouse bone marrow (BM) were estimated using the in vitro proliferative responsiveness [( 3H]thymidine incorporation) of the treated BM cells to recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human interleukin 2 (IL-2). From the response pattern of the treated BM cells to G-CSF and IL-2, the anticancer drugs were classified into three groups: (a) BM cells from cyclophosphamide- or nimustine hydrochloride-treated mice showed an increased responsiveness to G-CSF but a decreased responsiveness to IL-2; (b) BM cells from vindesine- or peplomycin-treated mice showed an increased responsiveness to both G-CSF and IL-2; and (c) BM cells from mitomycin C-treated mice showed a decreased responsiveness to both G-CSF and IL-2. These different response patterns may reflect qualitative differences in the myelotoxicity effects of these anticancer drugs.     

Long-term survival data from a phase 3 study of Filgrastim as an adjunct to chemotherapy in adults with de novo acute myeloid leukemia.

We previously reported the results of a double-blind, placebo-controlled study of Filgrastim in patients with de novo AML undergoing induction and consolidation chemotherapy. The study demonstrated that Filgrastim was effective and well tolerated and had no impact on complete remission or survival. We now report follow-up data on these patients, assessing long-term effects with emphasis on prognostic indicators. After a median follow-up of 7 years, 434 (83%) patients were dead, 73 (14%) were alive, and 14 (3%) were lost to follow-up. The proportions of deaths were similar in the Filgrastim (83%) and placebo (84%) groups. No differences in median time to death (1.04 years Filgrastim, 1.13 years placebo; P = 0.97) or median disease-free survival (0.86 years Filgrastim, 0.79 years placebo; P = 0.52) were evident. Proportional hazard modeling identified age, performance status, and French-American-British subtype as independent predictors for survival (P < 0.001, P = 0.005, and P = 0.036, respectively), whereas cytogenetic status was not (P = 0.118). Filgrastim had no effect on overall survival in any of these subgroup analyses as none of the treatment comparisons were statistically significant. These findings indicate that Filgrastim can be effectively used to support patients with AML undergoing induction and consolidation chemotherapy without worsening long-term disease outcome.     

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.     

G-CSF in the Biology and Treatment of Acute Myeloid Leukemias

Granulocyte colony-stimulating factor (G-CSF) is an hemopoietic growth factor produced by fibroblasts, monocytes and endothelial cells. The role of G-CSF in the biology of acute myeloid leukemia (AML) has been investigated by several authors, who have demonstrated receptor mediated enhanced proliferation of AML blasts in vitro, in the presence of G-CSF. This effect is further increased by addition of other cytokines such as GM-CSF, IL3, IL4, Stem cell factor (SCF), while Tumor Necrosis Factor (TNF) and Transforming Growth Factor β1 (TGF β1) seem to exert an inhibitory activity. An autocrine production of G-CSF by AML cells, a paracrine production by accessory cells and a protective effect displayed by G-CSF against programmed cell death could partially contribute to explain the pathogenesis of AML. In vivo, G-CSF has been used after chemotherapy in AML, in order to improve hemopoietic recovery in patients at high risk of infection. Current studies are focusing on better definition of the role of G-CSF, as such or combined with other biological modifiers, in dose intensification and autologous bone marrow or peripheral blood stem cell transplantation.     

Presently available biosimilars in hematology-oncology �?Part II: G-CSF

Biopharmaceuticals were copies of endogenous human proteins developed in the mid-nineties that were characterized by complex three-dimensional, high-molecularweight compounds. What made them unique was that contrary to classical chemotherapeutical drugs, they were manufactured by living cells. One of these biopharmaceuticals was granulocyte-colony stimulating factor (G-CSF). Once their patent expired, generic versions appeared in pharmacies. They are now called biosimilars. There are several biosimilar G-CSFs approved in Europe: Biograstim/Filgrastim ratiopharm/Ratiograstim/Tevagrastim (XM02); Zarzio and Nivestim. All these new products are manufactured in facilities with state-of-the-art technology. All products have passed the regulatory requirements for approval, mainly Phase I and Phase III, with the consequent PD/PK evaluations and studies on efficacy and safety. However, there are still someconcerns regarding their long-term evaluation, in particular, the limited experience at the time of approval of these products in terms of efficacy, safety and immunogenicity. For this reason, pharmacovigilance should be rigorous. A lot of work remains to be done in terms of clarification with regard to substituting a biosimilar G-CSF for the innovator product and, finally, information must be provided to physicians, pharmacists and patients to allow for proper decision making. Ultimately, only clinical trials and effective post-marketing pharmacovigilance will provide definitive evidence that a biosimilar is comparable to the originator-reference product in terms of efficacy and safety.