Recombinant human granulocyte colony-stimulating factor. Effects on hematopoiesis in normal and cyclophosphamide-treated primates.

We examined the in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in primates (cynomolgus monkeys) treated with subcutaneous doses of rhG-CSF for 14-28 d. A dose-dependent increase in the peripheral white blood cells (WBC) was seen, reaching a plateau after 1 wk of rhG-CSF treatment. The elevation of WBC was due to an increase in the absolute neutrophil count. These results demonstrate that rhG-CSF is a potent granulopoietic growth and differentiation factor in vivo. In cyclophosphamide (CY)-induced myelosuppression, rhG-CSF was able to shorten the time period of WBC recovery in two treated monkeys to 1 wk, as compared to more than 4 wk for the control monkey. Its ability to significantly shorten the period of chemotherapy-induced bone marrow hypoplasia may allow clinicians to increase the frequency or dosage of chemotherapeutic agents. In addition, the increase in absolute numbers of functionally active neutrophils may have a profound effect in the rate and severity of neutropenia-related sepsis. Furthermore, the activities reported here indicate a potential role for rhG-CSF in the treatment of patients with myelodysplastic syndrome, congenital agranulocytosis, radiation-induced myelosuppression, and bone marrow transplantation.

Identification and characterization of the protein encoded by the human N-myc oncogene.

The human N-myc gene is related to the c-myc proto-oncogene, and has been shown to have transforming potential in vitro. Many studies have reported amplification of N-myc in human neuroblastoma and retinoblastoma cell lines. In primary tumors, amplification of the gene was found to correlate directly with behavior of the tumor. Specific restriction fragments of a partial complementary DNA clone of N-myc from LA-N-5 human neuroblastoma cells were placed into a bacterial expression vector for the purpose of producing antigens representative of the N-myc protein. Rabbits immunized with these antigens produced antisera that recognized a protein of 62-64 kilodaltons in neuroblastoma cells. By several criteria, this protein appears to be part of the same proto-oncogene family as the c-myc protein. Moreover, the antisera to fragments of this protein were capable of histochemically identifying malignant cells in clinical specimens.

Studies of the human c-myb gene and its product in human acute leukemias.

The myb gene is the transforming oncogene of the avian myeloblastosis virus (AMV); its normal cellular homolog, c-myb, is conserved across a broad span of evolution. In humans, c-myb is expressed in malignant hematopoietic cell lines and in primary hematopoietic tumors. Partial complementary DNA clones were generated from blast cells of patients with acute myelogenous leukemia. The sequences of the clones were compared to the c-myb of other species, as well as the v-myb of AMV. In addition, the carboxyl terminal region of human c-myb was placed in an expression vector to obtain protein for the generation of antiserum, which was used to identify the human c-myb gene product. Like v-myb, this protein was found within the nucleus of leukemic cells where it was associated with the nuclear matrix. These studies provide further evidence that c-myb might be involved in human leukemia.

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells.

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases.

The balance between levels of metalloproteinases and their corresponding inhibitors is a critical factor in tumor invasion and metastasis. Down-regulation of the activity of these proteases was achieved by transfection of invasive and metastatic rat cells with the complementary DNA for metalloproteinase inhibitor/tissue inhibitor of metalloproteinase 2 (MI/TIMP-2), a novel inhibitor of metalloproteinases recently described. (Y. A. DeClerck et al., J. Biol. Chem., 264: 17445-17453, 1989; W. G. Stetler-Stevenson et al., J. Biol. Chem., 264: 17374-17378, 1989). Secretion of functional MI/TIMP-2 protein in stably transfected cells resulted in a marked decrease in metalloproteinase activity. Partial suppression of the formation of lung colonies after i.v. injection in nude mice was observed in a transfected clone expressing high levels of MI/TIMP-2. Production of MI/TIMP-2 in four clones markedly reduced tumor growth rate in vivo after s.c. injection and completely suppressed local tissue invasion. Thus, down-regulation of metalloproteinase activity has a striking effect on local invasion and partially suppresses hematogenous metastasis.

Expression patterns of immediate early transcription factors in human non-small cell lung cancer. The Lung Cancer Study Group.

In 1995, there will be 172,000 new cases of lung cancer diagnosed and 153,000 deaths from this disease in the United States. While the pathogenesis of the disease process is poorly understood, a growing body of evidence suggests that abnormalities in cellular regulatory genes may play an important role in the induction, maintenance and/or progression of some tumor types. These genes include both growth promoting oncogenes as well as growth inhibitory or suppressor genes. Included among these genetic sequences are several cellular transcription factors. A group of these factors including c-jun, c-fos and EGR1 are members of a class of genes known as immediate early genes whose expression are inducible by a variety of stimuli including mitogenic and differentiation inducing growth factors, indicating a potential important role for these genes in normal growth processes. Since these genes are involved in early regulation of cellular growth properties and at least two (c-jun and c-fos) can act as oncogenes, we wished to determine whether their expression levels were altered in human non-small cell lung cancers (NSCLC) compared to normal lung tissue. To address this, Northern blot analyses were performed using c-fos, c-jun and EGR1 probes on RNA extracted from 101 NSCLC tumor specimens and adjacent uninvolved lung tissue. Analysis of this cohort revealed that 72% of the normal tissues demonstrate significantly greater expression of these transcription factors as compared to adjacent malignant tissue. Moreover, this expression pattern appeared to be coordinate for all three genes in the majority of cases. This differential expression pattern was confirmed at the protein level using an immunohistochemical approach with antibodies directed against the c-jun, c-fos and EGR1 gene products. Southern blot analyses demonstrated no gross alterations of these sequences at the DNA level, indicating that the observed differential expression pattern was not due to gross structural changes in the genes. These data suggest that down-regulation of these genes may be involved in the pathogenesis of lung cancer.

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis in normal cats.

The objective of this study was to determine how recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects hematopoiesis in normal cats. Recombinant human G-CSF was given at 3.0, 5.0, and 10.0 micrograms/kg to two cats each s.c. twice daily for 21 days. This resulted in significant (p less than 0.01) elevations of peripheral blood neutrophils from 3.0- to 9.2-fold above pretreatment levels and significantly (p less than 0.02) above levels of nontreated control cats (n = 4). A statistically significant dose-related response was not seen at these dosages in any parameter evaluated. The period of maximum neutrophilia occurred between days 10 and 14 of rhG-CSF treatment, with maximum neutrophil counts ranging from 20,370 cells/microliters to 61,400 cells/microliters (normal is less than 12,500). Lymphocytosis (greater than 7000 lymphocytes/microliters) and monocytosis (greater than 850 monocytes/microliters) were observed in 50% of the cats receiving rhG-CSF during the period of maximal neutrophil stimulation. Monocyte counts in treated cats were significantly (p less than 0.01) elevated over those of treatment controls on days 12-17. Lymphocyte numbers in rhG-CSF-treated cats were significantly elevated (p less than 0.05) over pretreatment controls on days 12 and 14 of rhG-CSF treatment. No significant changes were observed in reticulocyte counts, platelet counts, or hematocrit levels. By day 19, neutrophil levels had dropped significantly (p less than 0.01) from the maximum neutrophil levels, with one cat attaining a normal blood neutrophil count by day 21 of rhG-CSF treatment. Marrow aspirates revealed an overall increase in marrow cellularity through day 14 of treatment in rhG-CSF-treated cats, with increased myeloid:erythroid ratios (two- to ninefold) over those of nontreated controls. The erythroid and lymphoid component of the marrow decreased from day 0 to day 14, whereas the early myeloid progenitors (myeloblasts, progranulocytes, and myelocytes) increased significantly (p less than 0.05). No significant differences in the percentage of later myeloid forms in the marrow were observed over the treatment period. In vitro colony-forming assays of marrow obtained from treated cats revealed increases in granulocyte-macrophage colony-forming units (CFU-GM) through day 14, with subsequent decreases by day 21 of rhG-CSF treatment. Recombinant human G-CSF was also effective at in vitro stimulation of feline marrow cells from untreated cats in a dilution study, with maximal CFU-GM formation at 0.1 microgram rhG-CSF/ml assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutrophil function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor.

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined. CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions. Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3. The drug was specific for neutrophils as determined by differential cell counts. Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo. Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E. coli. At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis. CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period. We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Increased granulopoiesis after sequential administration of transforming growth factor-beta 1 and granulocyte-macrophage colony-stimulating factor.

Transforming growth factor-beta 1 (TGF-beta 1) is an inhibitor of the growth and differentiation of immature hematopoietic progenitors in vitro; however, we have demonstrated that TGF-beta 1 can promote granulopoiesis in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. We therefore examined the effect of the combined administration of TGF-beta 1 and GM-CSF in vivo. First, TGF-beta 1 enhanced the specific binding of GM-CSF (2.0-fold) on bone marrow cells, reaching a maximum 40 hours after injection, while the specific binding of interleukin-3 (IL-3) was unaffected. Using GM-CSF-specific binding to determine the optimal regimen for cytokine administration in vivo, we found that the administration of TGF-beta 1 and GM-CSF in sequence increased myelopoiesis. Total numbers of colony-forming units-granulocyte/macrophage (CFU-GM) and myeloblasts per femur were increased above the level obtained with the simultaneous injection of TGF-beta 1 plus GM-CSF, GM-CSF alone or TGF-beta 1 alone. Further, the sequential administration of TGF-beta 1 and GM-CSF resulted in enhanced numbers of mature granulocytes in both the bone marrow and peripheral blood. In contrast, the sequential combination of TGF-beta 1 and GM-CSF did not enhance the numbers or increase the recovery of erythroid cells in the bone marrow. These results show that TGF-beta 1 in vivo as in vitro has a multifunctional effect on bone marrow progenitors, and by using an optimal combination of TGF-beta 1 and GM-CSF in vivo, one can selectively increase both the central and peripheral granulopoietic compartments.

Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber.

We have isolated the genomic and cDNA clones encoding a novel proteinase from the fungus Tritirachium album Limber, named proteinase T, synthesis of which is induced in skim milk medium. The coding sequence for this enzyme is interrupted by two introns in the fungal genome. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 53% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of two disulfide bonds, which might explain the thermal stability of the proteinase. We have expressed the proT cDNA in Escherichia coli. The authenticity of the product has been characterized by Western blotting and N-terminal analysis of the recombinant product.

Comparative analysis of the effects of recombinant cytokines on the growth and differentiation of ML-1, a human myelogenous leukemic cell line.

We have investigated the effect of a number of cytokines on the human acute myelomonocytic leukemic cell line, ML-1. The differentiation inducing effects of interleukins (IL-1 beta, IL-3 and IL-6), colony stimulating factors (GCSF and GMCSF), TNF, LIF and IFNg, were studied either individually or in combination. Criteria for monocytic differentiation were as follows: an increase in the percentage of cells reducing nitroblue tetrazolium (NBT) salt, an increase in the alkaline phosphatase activity as well as the appearance of macrophagic phenotype. Among all the cytokines tested, only TNF was found to induce differentiation and to inhibit growth of ML-1 cells. IL-3, IL-6, interferon gamma, GCSF and to a smaller extent IL-1 beta and GMCSF synergized the differentiation inducing activity of TNF.

Recombinant human granulocyte colony stimulating factor: molecular and biological characterization.

Human or rodent bone marrow treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a CFU-GM assay yield predominantly granulocytic colonies. The specificity for granulocyte progenitors in vitro is also demonstrated in vivo by a five- to six-fold elevation in hamster peripheral blood neutrophils. Other cell types (monocytes, lymphocytes and eosinophils) remain stable. Analysis of mRNA from the bladder carcinoma cell line 5637 (1A6) shows the predominant species of mRNA codes for a mature protein of 174 amino acids. A small fraction of the mRNA can code for an alternative form of hG-CSF containing additional three amino acids between positions 35 and 36.

Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin.

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period.

To determine if periparturient immunosuppression in dairy cattle might be due to an alteration in total numbers of percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparatum through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCD8, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4+ cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.

Use of recombinant canine granulocyte colony-stimulating factor to decrease myelosuppression associated with the administration of mitoxantrone in the dog.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (less than 3,000/uL) for 2 days in the dogs that received rcG-CSF, and for 5 days in the dogs that received only mitoxantrone. Four of five dogs not treated with rcG-CSF and none of those receiving rcG-CSF developed serious neutropenia (less than 1,500/uL). The neutrophil counts were significantly (P less than 0.05) higher in the rcG-CSF treated dogs at all time points except before the administration of the colony-stimulating factor, and the sixth day after the mitoxantrone was administered. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.

Effect of recombinant canine granulocyte colony-stimulating factor on peripheral blood neutrophil counts in normal cats.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously at a dosage of 5 micrograms/kg/day to five healthy, young adult cats for 42 days. Mean neutrophil counts +/- standard deviation increased significantly (P < 0.001) from 10,966/microL +/- 2324 to 30,688/microL +/- 5296 within 24 hours after administration of the first dosage of rcG-CSF. Mean neutrophil counts reached 52,978/microL +/- 11,207 on day 6, representing a second significant increase (P < 0.01) over the previous 5 days. Mean neutrophil counts continued to increase, reaching 66,994/microL +/- 12,419 on day 14, then remaining within a range of 66,994 to 87,839/microL throughout the remainder of the study. The maximum mean neutrophil count was 87,839/microL +/- 8,695 on day 42. Neutrophil counts remained high until the administration of recombinant canine granulocyte colony-stimulating factor was discontinued 42 days after initiation of therapy. Once the rcG-CSF administration was discontinued, neutrophil counts returned to pretreatment values within 5 days. There were no significant changes in numbers of any of the other cell lines. There was no clinically significant toxicosis associated with the administration of rcG-CSF.

Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor.

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.

cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases.

The purification and characterization of a metalloproteinase inhibitor (MI) from bovine aortic endothelial cells, and the demonstration that it is related to, but distinct from, tissue inhibitor of metalloproteinase (TIMP), have previously been reported [De Clerck, Y. A., Yean, T.-D., Ratzkin, B. J., Lu, H.S. & Langley, K. E. (1989) J. Biol. Chem. 264, 17445-17453]. The cDNA cloning of the bovine MI and its human homolog is now reported. The bovine cDNA cloning used probes designed on the basis of NH2-terminal amino acid sequence of bovine MI. The human cDNA cloning in turn used probes representing parts of the bovine cDNA nucleotide sequence. Both cDNAs encode leader sequences of 26 amino acids and mature protein sequences of 194 amino acids. The amino acid sequences of the mature proteins are 94% identical. The human MI cDNA was expressed in Escherichia coli, and a preparation containing anticollagenase activity was recovered. The amino acid sequence of mature human MI is 38% identical to the sequence for human TIMP, and the 12 cysteines in MI and TIMP are aligned almost identically. Thus MI and TIMP comprise an inhibitor family.