Nestin expression--a property of multi-lineage progenitor cells?

Tissue-specific progenitor cells are characterized by proliferation and differentiation, but, in contrast to embryonic stem (ES) cells, have limited capacities for self-renewal and no tumourigenic potential. These latter traits make progenitor cells an ideal source for regenerative cell therapies. In this review, we describe what is currently known about nestin, an intermediate filament first identified in neuroepithelial stem cells. During embryogenesis, nestin is expressed in migrating and proliferating cells, whereas in adult tissues, nestin is mainly restricted to areas of regeneration. We show that nestin is abundant in ES-derived progenitor cells that have the potential to develop into neuroectodermal, endodermal and mesodermal lineages. Although it remains unclear what factors regulate in vitro and in vivo expression of nestin, we conclude that nestin represents a characteristic marker of multi-lineage progenitor cells and suggest that its presence in cells may indicate multi-potentiality and regenerative potential.

Doublet discrimination in DNA cell-cycle analysis.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult.

Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity.

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-1-phosphate (S1P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S1P-induced endothelial cell migration was maximum at 1 microM (approximately 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) (approximately 2.5-fold increase) or hepatocyte growth factor (HGF) (approximately 2.5-fold increase). Migration induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial differentiation gene) S1P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a betagamma subunit inhibitory peptide of the beta-adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G(1alpha2) signaling. Various strategies to interrupt Rho family signaling, including C(3) exotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S1P- but not VEGF-induced migration. Conversely, pharmacologic inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase reduced basal endothelial cell migration and abolished VEGF-induced endothelial cell migration but did not inhibit the increase in S1P-induced migration. Whereas VEGF and S1P increased both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein (MAP) kinase activities, only p38 MAP kinase inhibition significantly reduced VEGF- and S1P-stimulated migration. These data confirm S1P as a potent endothelial cell chemoattractant through G(1alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 MAP kinase activation. The divergence in signaling pathways evoked by S1P and VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.

Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques.

Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.

Fibronectin fragment CH-296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human lentivirus vectors independent of viral tropism in nonhuman primate CD34+ cells.

The fibronectin fragment CH-296 improved gene transfer to cytokine-mobilized nonhuman primate CD34+ cells irrespective of tropism to the MoMLV, GaLV, and VSV-G envelope proteins using murine stem cell virus (MSCV) and human immunodeficiency virus-1 (HIV-1)-based retrovirus vectors. For the HIV-1 lentivirus vector, CH-296 enhanced gene transfer in the absence of added hematopoietic growth factors necessary for ex vivo stem cell expansion. In the presence of CH-296, apoptosis of CD34+ cells was inhibited, and in mobilized peripheral blood CD34+ cells, cell division was stimulated as measured by cell history/tracking experiments.

Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells.

Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.

High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)

Transplantation of transduced nonhuman primate CD34+ cells using a gibbon ape leukemia virus vector: restricted expression of the gibbon ape leukemia virus receptor to a subset of CD34+ cells.

The transduction efficiencies of immunoselected rhesus macaque (Macaca mulatta) CD34+ cells and colony-forming progenitor cells based on polymerase chain reaction (PCR) analysis were comparable for an amphotropic Moloney murine leukemia virus (MLV) retroviral vector and a retroviral vector derived from the gibbon ape leukemia virus (GaLV) packaging cell line, PG13. On performing autologous transplantation studies using immunoselected CD34+ cells transduced with the GaLV envelope (env) retroviral vector, less than 1% of peripheral blood (PB) contained provirus. This was true whether bone marrow (BM) or cytokine-mobilized PB immunoselected CD34+ cells were reinfused. This level of marking was evident in two animals whose platelet counts never fell below 50,000/microliter and whose leukocyte counts had recovered by days 8 and 10 after having received 1.7 x 10(7) or greater of cytokine-mobilized CD34+ PB cells/kg. Reverse transcriptase(RT)-PCR analysis of CD34+ subsets for both the GaLV and amphotropic receptor were performed. The expression of the GaLV receptor was determined to be restricted to CD34+ Thy-1+ cells, and both CD34+ CD38+ and CD34+ CD38dim cells, while the amphotropic receptor was present on all CD34+ cell subsets examined. Our findings suggest that, in rhesus macaques, PG13-derived retroviral vectors may only be able to transduce a subset of CD34+ cells as only CD34+ Thy-1+ cells express the GaLV receptor.

Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins.

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (DeltaE1) or lacking the Ad E3 region in addition to E1 sequences (DeltaE1DeltaE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34(cdc2) protein levels. In some instances, infection with DeltaE1 or DeltaE1 DeltaE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DeltaE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in DeltaE1 or DeltaE1 DeltaE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.

Thapsigargin-induced gene expression in nonexcitable cells is dependent on calcium influx.

Agents such as thapsigargin and endothelin elevate intracellular calcium levels by a combination of calcium release from intracellular stores and calcium influx across the plasma membrane; however, the relative contribution of influx vs. release in modulating calcium-dependent gene expression is not as well understood in nonexcitable cells as in excitable cells. In this report we have been able to separate thapsigargin-induced elevation of intracellular calcium into release and influx components, using carboxyamido-triazole (CAI), a known inhibitor of calcium influx with antiproliferative activity against a number of human carcinomas, to selectively inhibit influx without affecting release. The results of these experiments indicate that the ability of thapsigargin to induce calcium-dependent gene expression in nonexcitable cells is dependent on the induction of calcium influx, presumably through store-operated calcium channels. CAI treatment specifically inhibited thapsigargin- or endothelin-stimulated expression from the c-fos promoter in Rat-1 cells and in epithelial cell lines derived from ovary and breast. Use of the VL30 model system confirmed the ability of CAI to inhibit calcium-dependent gene expression and further demonstrated that the ability of elevated calcium to synergize with other signaling pathways required close temporal coupling. In addition to inhibiting endothelin-induced calcium influx, CAI treatment also resulted in a partial inhibition of IP3 production and calcium release. CAI treatment also blocked the increase in ERK1 kinase activity observed in response to either endothelin or thapsigargin, suggesting a role for calcium influx in the activation of mitogen-activated protein kinase pathways.

Identification and characterization of human metabolites of CAI [5-amino-1-1(4'-chlorobenzoyl-3,5-dichlorobenzyl)-1,2,3-triazole- 4-carboxamide).

The calcium influx inhibitor and cytostatic agent, 5-amino-1-1(4'-chlorobenzoyl-3,5-dichlorobenzyl)-1,2,3-triazole-4-carboxamide (CAI), is in phase I clinical trial for patients with refractory cancer. Additional chromatography peaks were observed during HPLC analysis of patient samples. Identification and characterization of physiological metabolites were undertaken using HPLC techniques developed for their purification from blood, pleural fluid, and urine samples. A hydrophobic metabolite, M1, was purified and functionally characterized. Structural analysis of the purified compound indicated that it is a 3,5-dichloro-4(p-chlorobenzoyl)-benzoic acid. Quantitative analysis of M1 concentration during CAI administration indicated that the rise in M1 concentration lagged behind that of CAI and persisted after CAI was no longer detectable. No clear relationship between CAI or M1 and either toxicity or efficacy was observed. Chromatography of patient blood and urine samples under conditions favoring hydrophilic metabolite detection suggested the presence of a glucuronide compound; this was also indicated by sample treatment with beta-glucuronidase. Attempts at purification did not yield a compound stable for structural analysis. The benzophenone metabolite, M1, was nonfunctional in assays of calcium influx inhibition or proliferation. No pharmacodynamic associations were observed for these metabolites, nor was there pharmacological activity of the M1 as an individual agent. These data suggest that CAI is processed into triazole and benzophenone moieties by phase I metabolism, and these metabolites or the parent compound may be conjugated for excretion by glucuronidation.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Debris compensation of DNA histograms and its effect on S-phase analysis.

Debris compensation is an important variable affecting S-phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP-SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC-MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult.

Angiogenesis: role of calcium-mediated signal transduction.

During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.

Direct in vivo gene transfer and expression in malignant cells using adenovirus vectors.

To evaluate the ability of replication-deficient, recombinant adenovirus vectors to transfer genes to human tumor cells in vivo, adenovirus vectors containing the Escherichia coli lacZ (Ad.RSV beta gal) gene (coding for beta-galactosidase; used as a cell marker for gene transfer) or the human alpha 1-antitrypsin (Ad-alpha 1AT) cDNA (used as an example of a secreted protein) were administered intraperitoneally to nude mice with human malignant mesothelioma cell (H-MESO-1) malignant ascites. Preliminary in vitro studies showed that both vectors effectively transferred genes to H-MESO-1 cells. Tumor cells recovered from ascites of animals intraperitoneally administered a control adenovirus revealed no evidence of beta-galactosidase (beta-gal) activity 3 or 14 days later. In contrast, beta-gal activity was detected at the same time points in tumor cells from animals receiving intraperitoneal Ad.RSV beta gal. Flow cytometric quantification of beta-gal activity in recovered cells showed < 3% beta-gal-positive cells in animals administered control virus, but in animals administered intraperitoneal Ad.RSV beta gal there was a mean of 71 +/- 18% positive cells at 3 days and 56 +/- 27% at 14 days. Human alpha 1AT was not detected by enzyme-linked immunosorbent assay (ELISA) in ascites of animals receiving a control virus; however, in ascites of animals administered Ad-alpha 1AT, 21,000 +/- 3,800 ng/ml of human alpha 1AT was detected at 3 days and 4,900 +/- 1,700 ng/ml at 14 days. These data demonstrate that replication-deficient recombinant adenovirus vectors can be used to transfer genes to malignant cells in vivo and suggest a new strategy for genetic modification for antitumor therapy.

Asymmetric distribution of oncogene products at mitosis.

Computer-assisted image analysis was used to demonstrate in exponentially proliferating human tumor cells the uneven postmitotic apportionment of several oncogene-encoded proteins (ras p21; erbB-2 p185; fos p55; myc p62). This observation may provide the explanation for the high degree of heterogeneity of postmitotic cells and the asynchrony in cell cycle traverse of cultured cells.

Flow cytometric DNA analysis of human solid tumors: a review of the interpretation of DNA histograms.

A survey of over 225 recent studies examining the relationship between the flow cytometric DNA analysis of solid tumors and clinical prognosis indicates that criteria used to classify DNA histograms are variable and often inconsistent with the recommendations proposed by the Convention on Nomenclature for DNA Cytometry. Numerous reports not only lack unambiguous descriptions of the histogram features used to differentiate diploid from aneuploid DNA distributions, but also inadequately describe the technical aspects of data acquisition, standardization, and inclusion or exclusion of subpopulations by gating. In many cases, the coefficient of variation of the diploid and aneuploid G0/1 peaks, which would allow an assessment of histogram quality, is not reported. Because of the differences in DNA histogram interpretation, extrapolation of the results among laboratories may be difficult and is probably not reliable. This review summarizes the criteria that have been used to classify the DNA histograms and illustrates the effects of these different classifiers on DNA ploidy analysis and clinical conclusions.

Protein expression in relation to the cell cycle of exponentially growing human prostatic epithelial cells.

This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.