Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 µm for surfaces with small pillar sizes of 1 and 2 µm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 µm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Interleukin-4-mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages.

Upon interferon-gamma (IFN-gamma) stimulation, murine macrophages (Mphi) produce nitric oxide (NO) through expression of inducible nitric oxide synthase (iNOS). Interleukin (IL)-4 treatment, even delayed 12 h relative to IFN-gamma, antagonized this induction, whereas infection with herpes simplex virus type 2 (HSV-2) or treatment with tumour necrosis factor-alpha exerted a synergistic effect, which partly compensated for the antagonistic effect of IL-4. Neither IL-4 nor HSV-2 affected the IFN-gamma-activated Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway or altered the levels of IFN-gamma-induced interferon regulatory factor (IRF)-1 expression, which is STAT1-dependent and known to play a central role in IFN-gamma-mediated gene induction. The effect of IL-4 was completely dependent on de novo protein synthesis, indicating that a direct activation of latent inhibitors is not sufficient to explain the inhibitory effect of IL-4. Furthermore, IL-4 substantially augmented the IFN-gamma-induced expression of IRF-2, which is known to compete with IRF-1 for the DNA recognition site, ISRE (interferon-stimulated response element). Our findings could indicate that IL-4 suppresses IFN-gamma-stimulated iNOS transcription by elevating the level of IRF-2 which, through competition, prevents IRF-1 from binding to ISRE in the iNOS promoter. The virus-induced effects on iNOS and NO levels in IFN-gamma-stimulated Mphi do not seem to involve the Jak/STAT pathway or a differential expression of IRF-1 and IRF-2.

B-Cell lymphoma induction by akv murine leukemia viruses harboring one or both copies of the tandem repeat in the U3 enhancer.

Akv is an endogenous, ecotropic murine leukemia virus (MuLV) of the AKR strain. It has served as a prototype nonpathogenic or weakly pathogenic reference virus for studies of closely related potent lymphomagenic viruses such as the T-lymphomagenic SL3-3. We here report that Akv and an Akv mutant (Akv1-99) with only one copy of the 99-bp transcriptional enhancer induce malignant lymphomas with nearly 100% incidence and mean latency periods of 12 months after injection into newborn NMRI mice. Molecular analysis of tumor DNA showed that the majority of the tumors were of the B-cell type. Sequence analysis of proviral transcriptional enhancers in DNA of B-cell lymphomas revealed conservation of the enhancer sequence, as well as a lack of sequence duplications of the Akv1-99 variant, while the repeat copy number in Akv was subject to fluctuations. In support of a B-cell specificity of the Akv enhancer, a murine plasmacytoma cell line was found to sustain three- to fivefold-higher transient transcriptional activity upon the Akv and Akv1-99 enhancers than upon the enhancer of the T-lymphomagenic SL3-3 MuLV. Thus, the overall picture is that Akv MuLV possesses a B- lymphomagenic potential and that the second copy of the 99-bp sequence seems to be of minor importance for this potential. However, in one animal the lymphomas induced by Akv1-99 were of the T-cell type. Among the 24 tumors analyzed only this one harbored a clonal proviral integration in the c-myc locus. This provirus had undergone a duplication of a 113-bp sequence of the enhancer region, partly overlapping with the 99-bp repeat of Akv, as well as a few single nucleotide alterations within and outside the repeats. Taken together with previous studies, our results suggest that T- versus B-lymphomagenic specificity of the enhancer is governed by more than one nucleotide difference and that alterations in binding sites for transcription factors of the AML1 and nuclear-factor-1 families may contribute to this specificity.

Increased lymphomagenicity and restored disease specificity of AML1 site (core) mutant SL3-3 murine leukemia virus by a second-site enhancer variant evolved in vivo.

SL3-3 is a highly T-lymphomagenic murine retrovirus. The major genetic determinant of disease is the transcriptional enhancer, which consists of a repeated region with densely packed binding sites for several transcription factors, including AML1 (also known as core binding factor and polyoma enhancer-binding protein 2) and nuclear factor 1 (NF1). Previously, we examined the enhancer structure of proviruses from murine tumors induced by SL3-3 with mutated AML1 (core) sites and found a few cases of second-site alterations. These consisted of deletions involving the NF1 sites and alterations in overall number of repeat elements, and they conferred increased enhancer strength in transient transcription assays. We have now tested the pathogenicity of a virus harboring one such second-site variant enhancer in inbred NMRI mice. It induced lymphomas with a 100% incidence and a significantly shorter latency than the AML1 mutant it evolved from. The enhancer structure thus represents the selection for a more tumorigenic virus variant during the pathogenic process. Sequencing of provirus from the induced tumors showed the new enhancer variant to be genetically stable. Also, Southern blotting showed that the tumors induced by the variant were T-cell lymphomas, as were the wild-type-induced lymphomas. In contrast, tumors induced by the original core/AML1 site I-II mutant appeared to be of non-T-cell origin and several proviral genomes with altered enhancer regions could be found in the tumors. Moreover, reporter constructs with the new tumor-derived variant could not be transactivated by AML1 in cotransfection experiments as could the wild type. These results emphasize the importance of both core/AML1 site I and site II for the pathogenic potential of SL3-3 and at the same time show that second-site alterations can form a viral variant with a substantial pathogenic potential although both AML1 sites I and II are nonfunctional.

Effect of IL-4 and IL-13 on IFN-gamma-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2.

Interleukin (IL)-4 and IL-13 share a wide range of activities. Prominent among these is the ability to antagonize many interferon (IFN)-gamma-induced activities. Here we demonstrate that IL-4 and IL-13 totally abrogate IFN-gamma-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA and protein synthesis in a murine macrophage cell line. IFN-gamma-treated cells infected with herpes simplex virus type 2 (HSV-2) or costimulated with tumor necrosis factor (TNF)-alpha showed an enhanced reactivity, which was only partially reduced by IL-4/13. The results indicate that IL-4 and IL-13 function by intervening with a step prior to iNOS transcription by antagonizing IFN-gamma-induced signal(s) without counteracting synergistic virus- or TNF-alpha-induced signals. The beneficial effect of a sustained NO production in foci of virus infection is suggested.

Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site.

SL3-3 is a highly T-lymphomagenic murine retrovirus. Previously, mutation of binding sites in the U3 repeat region for the AML1 transcription factor family (also known as core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF1]) were found to strongly reduce the pathogenicity of SL3-3 (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström, J. Virol. 65:4177-4181, 1991). We have now examined the few cases in which tumors developed harboring proviruses that besides the AML1 (core) site mutations carried second-site alterations in their U3 repeat structures. In three distinct cases we observed the same type of alteration which involved deletions of regions known to contain binding sites for nuclear factor 1 (NF1) and the addition of extra enhancer repeat elements. In transient-expression experiments in T-lymphoid cells, these new U3 regions acted as stronger enhancers than the U3 regions of the original viruses. This suggests that the altered proviruses represent more-pathogenic variants selected for in the process of tumor formation. To analyze the proviral alterations, we generated a series of different enhancer-promoter reporter constructs. These constructs showed that the additional repeat elements are not critical for enhancer strength, whereas the NF1 sites down-regulate the level of transcription in T-lymphoid cells whether or not the AML1 (core) sites are functional. We therefore also tested SL3-3 viruses with mutated NF1 sites. These viruses have unimpaired pathogenic properties and thereby distinguish SL3-3 from Moloney murine leukemia virus.

Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer.

Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.

Herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha.

We have analysed the ability of herpes simplex virus type 2 (HSV-2) to induce nitric oxide (NO) production in resting BALB/c mouse peritoneal macrophages. In most experiments, macrophages produced very small amounts of NO upon infection with HSV-2. Mock virus preparations did not induce NO production, and virus inactivation experiments showed that infectious virus was required. Since interferon-gamma (IFN-gamma) is the prototype cytokine that is able to induce significant NO production in macrophages, we found it of interest to examine the influence of HSV-2 infection on the IFN-gamma-induced NO production. The virus exerted a synergistic effect on the IFN-gamma-induced NO release, which was accompanied by induction of the iNOS-gene as revealed by RT-PCR. This effect was largely dependent on the presence of infectious virus particles, since only a minor effect was seen with mock virus and inactivated virus preparations. From experiments with neutralizing antibodies to tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha/beta it was concluded that the synergistic effect is dependent on autocrine secretion of TNF-alpha, which acts as a second signal and synergizes with IFN-gamma in NO production.

Mapping of a major osteomagenic determinant of murine leukemia virus RFB-14 to non-long terminal repeat sequences.

Certain isolates of murine leukemia viruses (MuLVs) have, apart from a leukemogenic potential, the capability of inducing diseases of nonhematopoietic tissues in susceptible strains of mice. We have reported on the molecular cloning of a bone-tumorigenic virus, RFB-14 MuLV, which was found to induce benign bone tumors, osteomas, with 100% incidence in mice of the CBA/Ca strain (L. Pedersen, W. Behnisch, J. Schmidt, A. Luz, F. S. Pedersen, V. Erfle, and P. G. Strauss, J. Virol. 66:6186-6190, 1992). In order to analyze the bone tumor-inducing phenotype of RFB-14 MuLV, we have studied the pathogenic potential of recombinant viruses between RFB-14 and the nonosteomagenic, highly leukemogenic SL3-3 MuLV. The recombinants were constructed so as to reveal whether a major determinant of osteomagenicity maps to sequences within or outside the long terminal repeats (LTR). Our data show that a major determinant of the osteoma-inducing potential of RFB-14 MuLV maps to the non-LTR region of the genome. Furthermore, we demonstrate that a strong determinant of leukemogenicity is harbored by the non-LTR region of SL3-3 MuLV.

The effect of selection for high-level vector expression on the genetic and functional stability of a single transcript vector derived from a low-leukemogenic murine retrovirus.

Single-gene murine leukemia virus-based retroviral vectors carrying the G418-resistance gene (neo) under transcriptional control of the long terminal repeat were used to study the effect of selection on long-term vector expression in a murine lymphoid cell line, L691. We used two isogenic vectors carrying either a strong or a weak transcriptional enhancer from low-leukemogenic Akv and high-leukemogenic SL3-3 murine leukemia virus, respectively. Effects of G418 selection were studied at the level of vector-transduced cell populations and at the level of single-vector-transduced cell clones obtained without selection for vector expression. Selection for vector expression prior to isolation of cell clones changed the range of vector expression for the two populations of cell clones. Cell clones harboring the Akv enhancer, isolated without selection and then subjected to prolonged growth under selective conditions, exhibited no mutations in the enhancer region or major vector rearrangements although showing increased vector expression in some cases. Our results are discussed in terms of retrovirus-mediated gene transfer strategies employing selection for expression of a selective marker in single-gene or bicistronic vectors with a low- or nonleukemogenic virus-derived backbone.

Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication.

Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching tRNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site.

A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses.

Identical transcription units inserted at different positions of mammalian chromosomes may vary widely in transcriptional activity. We have used a set of ten cell clones with random unselected single integrations of retroviral vectors to study such position effects. The vector used carries a neo gene driven by the Akv murine leukemia virus long terminal repeat that has only a weak promoter-enhancer activity in the target cell, the lymphoid cell line L691. Under transient expression conditions, the strength of the Akv promoter-enhancer in the L691 cells is increased by dexamethasone. In cell clones with single vector integrations, a correlation is observed between the non-induced expression levels and the degree of dexamethasone induction. The strongest relative induction is found for the integrated vectors with the lowest non-induced expression levels and approaches the inducibility under transient expression. These results indicate that expression levels are composed of distinct contributions from the integrated vector and from the site of integration and are best explained in terms of a model in which the sites of chromosomal integration exert variable positive enhancer effects upon vector transcription.

Involvement of nuclear factor I-binding sites in control of Akv virus gene expression.

The U3 region of Akv murine leukemia virus carries a 99-base-pair repeat that is associated with transcriptional enhancement in murine NIH 3T3 cells. Deletion analysis points to a critical function of a region within the repeat unit related to the recognition sequences for nuclear factor I proteins but distinct from the sites previously analyzed in related viruses. Nuclear proteins binding to the critical site were detected in NIH 3T3 cells and in mouse livers. A protein fraction binding to this site was purified from mouse livers by ion-exchange and DNA affinity chromatography and shown to have nuclear factor I properties. Mutations that caused a partial or complete reduction of the in vitro binding were introduced into an Akv long terminal repeat with one 99-base-pair repeat copy driving a reporter gene, and the expression activities of the mutants in NIH 3T3 cells were found to correspond to their in vitro binding activities. This correlation strongly supports the role of nuclear factor I proteins in Akv expression. Residual expression activity was, however, detected in mutants devoid of in vitro binding. This residual activity may relate to the presence of additional sequences with homology to nuclear factor I binding sites both within and outside the repeat region. The ability of these sites to bind crude and purified protein fractions with nuclear factor I activity was analyzed, and the role of the sites within and outside the repeat region for control of gene expression of Akv and related viruses is discussed.

Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon-alpha in epithelial, fibroblast, and lymphoid cells.

In all, 40 major polypeptides ranging in molecular weights from 14.5 to 83 kDa were shown to be induced by IFNs alpha (also by IFN-alpha 2b and beta in a few cases) and gamma in human cultured cells of epithelial (transformed amnion cells (AMA)), fibroblast (proliferating and quiescent MRC-5 fibroblasts), and lymphoid origin (Molt-4). With the exception of a heat shock protein (IEF14 or hs x 70) and two tropomyosins (IEFs 52x and 55), none of these proteins corresponded to polypeptides (proliferation-sensitive or others) previously identified and catalogued by us. IFN-alpha induced the highest number of polypeptides in lymphoid cells, while the response to IFN-gamma was more pronounced in cultured epithelial and fibroblast cells. Several of the polypeptides induced by IFNs alpha and gamma were synthesized (albeit at different rates) by the control untreated cells, and in some cell types such as normal human peripheral blood mononuclear cells many were expressed at high levels. Only IFN-alpha-induced a unique set of proteins (alpha 1, 51 kDa; alpha 2, 15 kDa; alpha 19, 78 kDa; and gamma 10, 83 kDa) in all cultured cell types studied, implying that response to this IFN involves a shared biochemical pathway(s). Both IFN-alpha (also IFN-alpha 2b) and beta induced an identical group of proteins in AMA cells in agreement with the fact that type I IFNs share common receptors. IFNs alpha and gamma induced a few common polypeptides, but only gamma 10 (83 kDa) showed increased synthesis in all cell types exposed to either of these IFNs. A total of 28 major cellular polypeptides were down-regulated by IFNs in the various cell type studied. Different sets of proteins were affected, however, in each system, emphasizing the complexity of the mechanisms underlying the action of these factors. Treatment of synchronized G1 AMA cells with IFNs alpha, beta, or gamma (500 IU/ml, final concentration) did not inhibit their progression from G1 to S-phase as determined by indirect immunofluorescence using PCNA autoantibodies specific for cyclin. These observations were in line with the fact that IFNs did not affect dividin or cyclin(PCNA) synthesis (S-phase specific proteins) at least within the first 17 hr after their addition.