Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential.

To examine the role of the Notch ligand Delta-4 on hematopoietic stem cells, human CD34+CD38low cord blood cells were cocultured on S17 cells transduced with transmembrane Delta-4 (mbD4/S17) or an empty vector (C/S17). By the end of a 3-week culture, mbD4/S17 induced a 25-fold reduction in nucleated cell production, as compared to C/S17, by maintaining a higher proportion of cells in G0/G1 phase. A specific retention of a high proportion of CD34+ cells throughout the culture was observed with mbD4/S17, contrary to C/S17. Although mbD4/S17 promoted expansion of cells with the phenotype of committed lymphoid precursors (CD34+CD7+CD45RA+), these cells still retained their myeloid differentiation potential. mbD4/S17 maintained a higher LTC-IC frequency in output CD34+ cells, compared to C/S17, as in the subsets of cells having completed the same number of divisions on mbD4/S17. A Delta4-Fc protein (extracellular part of human Delta4 fused to Fc human IgG1 portion), immobilized on plastic, also reduced cell production and retained the LTC-IC potential. Transplantation of cells grown on mbD4/S17 into NOD/SCID mice showed no significant enhancement of the long-term repopulating ability. Thus, Delta4 appears to inhibit hematopoietic stem cell proliferation, in association with the maintenance of short-term lymphoid and myeloid repopulation capacity.

Effects of various treatments of CFU-S seeding efficiency in mice.

Stem cell seeding efficiency and the proportion of CFU-S in the S phase after cytosine arabinoside (Ara C) treatments were assessed. We found no difference in the "f" factor between quiescent and cycling CFU-S. A survey of the literature indicates conflicting data regarding the possible correlation between the kinetic status of CFU-S and the "f" factor, but there is no question that the "f" factor can vary with treatment of either the donor or the recipient. It seems, therefore, important to determine the "f" factor for each new treatment protocol regardless of the cycling state of the stem cells if one is interested in knowing the absolute number of CFU-S in the marrow.

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. II. Unresponsiveness of isolated adult rat hepatocytes, 3T3, FDC-P2, and K562 cell lines to AcSDKP. Possible involvement of intermediary cell(s) in the mechanism of AcSDKP action.

Because the molecular mechanisms of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP; an inhibitor of spleen colony-forming unit [CFU-S] DNA synthesis) are difficult to study on bone marrow due to the scarcity of CFU-S in this tissue, we sought a pure cell population responsive to the molecule in vitro. Although growth factor-stimulated DNA synthesis in primary culture of hepatocytes and Balb/c 3T3 cells can be inhibited by transforming growth factor beta (TGF beta) and interferon alpha/beta (IFN[alpha/beta], respectively, neither hepatocytes nor 3T3 cells were found to be sensitive to AcSDKP. DNA synthesis in stimulated murine FDC-P2 cell lines and in human K562 cell lines also remained unchanged after exposure to the tetrapeptide. The fact that hepatocytes do respond in vivo to AcSDKP implies the existence of intermediary cell(s) involved in AcSDKP action in vivo that are lacking in hepatocyte culture. Whether intermediary cell(s) are implicated in the inhibitory action of AcSDKP on CFU-S entry into DNA synthesis is now being investigated.

Autocrine interferon-beta synthesis for gene therapy of HIV infection: increased resistance to HIV-1 in lymphocytes from healthy and HIV-infected individuals.

OBJECTIVE: To explore the possibility of gene therapy of HIV infection based on the multiple antiretroviral activities of interferon (IFN)-beta. DESIGN: We introduced into HIV target cells an IFN-beta gene placed under an expression control ensuring a low and constitutive expression, sufficient to confer a permanent antiviral state without impeding normal cell function. METHODS: We transformed, with an efficacy ranging from 20-55%, peripheral blood lymphocytes (PBL) derived from healthy, seronegative donors, and from asymptomatic HIV-infected individuals by the HMB-KbHuIFN beta retroviral vector carrying the human IFN-beta coding sequence driven by a fragment of the murine H-2Kb gene promoter. RESULTS: The replication rate of the IFN-beta-expressing cells was no different from that of untransformed controls during the 21-day period of in vitro observation. When IFN-beta-transformed, purified CD4+ lymphocytes from healthy donors were HIV-1LAI-infected, virus replication was inhibited and most of the cells survived, in contrast to untransformed CD4+ cells which were all destroyed 12 days after infection. Protection of CD4+ cells from the same donors was also observed in suspensions of IFN-beta-transformed total PBL that were infected with HIV-1LAI. In IFN-beta-transformed PBL from four HIV-infected donors, endogenous HIV replication was decreased and 28-69% of the CD4+ cells survived at the end of the 21 days in culture. In the untransformed control PBL suspensions, all CD4+ cells were destroyed. In long-term experiments, HIV-infected, IFN-beta-transformed cell populations of the lymphocytic CEM and the promonocytic U937 line were kept in culture for 60 days, during which time they remained resistant to HIV infection. CONCLUSION: These results indicate that further exploration of autocrine IFN-beta production for somatic cell gene therapy of HIV infection is warranted.

Development of methods for somatic cell gene therapy directed against viral diseases, using retroviral vectors carrying the murine or human interferon-beta coding sequence: establishment of the antiviral state in human cells.

We are developing methods for somatic cell gene therapy directed against chronic and fatal virus infections, such as acquired immunodeficiency (AIDS), by transforming cells with a constitutively expressed interferon (IFN) coding sequence. Previous work from our laboratory has shown that stable antiviral expression (SAVE) can be obtained in murine BALB/c 3T3 cells and human U937 cells transformed with plasmids carrying either the murine or the human IFN-beta coding sequence placed under the expression control of a 0.6-kb Xho II-Nru I promoter region of the murine H-2Kb major histocompatibility complex (MHC) gene (Macé et al., 1991; Seif et al., 1991). In the present paper, we report the construction of murine (Mu) and human (Hu) IFN-beta-expressing retroviral vectors (pMPZen-MuIFN beta, pHMB-KbMuIFN beta) and the problems encountered. Because of the murine origin of commonly used packaging cells and the species specificity of IFN, it was evident that placing the murine IFN-beta sequence under constitutive expression control could result in the production of Mu IFN in the murine packaging system, and thereby lead to decreased vector production and also to enhanced resistance of target cells. Using a packaging cell line that releases a beta-galactosidase-expressing vector, we show that, as expected, Mu IFN-alpha/beta decreases vector production of murine packaging cells and also inhibits the transformation of target NIH-3T3 cells with this vector, but the presence of anti-Mu IFN antibodies rescues the viral titer of the packaging cells and restores the sensitivity of target cells to virus transformation. However, the same antibody treatment is unable to rescue the viral titer of psi-2 packaging cells producing autocrine Mu IFN-beta encoded by the pMPZen-MuIFN beta and pHMB-KbMuIFN beta vectors. Because of the species specificity of IFN, this problem is circumvented with the pMFG-HuIFN beta vector carrying the human IFN-beta sequence. In spite of the production of Hu IFN, murine psi-CRIP packaging cells are able to release retroviral vectors expressing Hu IFN-beta, and these amphotropic vectors can transform human MRC-5 cells and confer to these cells an enhanced resistance to vesicular stomatitis virus (VSV) infection.

Macaque lymphocytes transduced by a constitutively expressed interferon beta gene display an enhanced resistance to SIVmac251 infection.

We are developing a method of gene therapy of HIV infection based on the low constitutive expression of an interferon beta (IFN-beta) gene in HIV target cells. Herein we report the first step in the development of a relevant animal model, provided by the macaque (Macaca fascicularis) infected with a pathogenic SIVmac251 isolate. To avoid the possibility of in vivo rejection of macaque lymphocytes expressing Hu IFN-beta, we have PCR-amplified and sequenced the Ma IFN-beta-coding sequence, and placed it under the control of a PstI-NruI 0.6-kb fragment of the murine H-2Kb gene promoter in the MFG-K(b)MaIFNbeta retroviral vector. Lymphocytic CEMX174 cells, transduced by coculture on packaging cells with this construct, harbored a mean of 0.07 to 1.2 copies of the IFN-beta transgene per cell, and were characterized by an IFN production ranging from 75 to 750 units per 5 x 10(5) cells per 3 days. The IFN-beta-transduced populations displayed an enhanced resistance against the pathogenic SIVmac251 isolate. Control experiments showed that the enhanced resistance could not be ascribed to the Ma IFN-beta released during the 3 days of coculture by the packaging cells, or to the mere transduction with a retroviral vector. Macaque lymphocytes transduced by the MFG-K(b)MaIFNbeta retroviral vector by coculture on packaging cells, acquired a mean number of IFN-beta transgene copies per cell ranging from 0.03 to 0.1. Such transduction led to the release of IFN-beta into the culture medium, ranging from 10 to 20 units per 5 x 10(5) cells per 3 days. This increased the anti-SIV resistance of the lymphocytes, as demonstrated by a decreased p27 antigen release into the culture medium, without affecting lymphocyte proliferation.

Antiviral activity of autocrine interferon-beta requires the presence of a functional interferon type I receptor.

We and others have previously observed that the antiviral effects of autocrine interferon (IFN)-alpha/beta activity cannot be abolished by neutralizing antibodies, even when present to a large excess. This raises the possibility that the major part of autocrine activity is triggered intracellularly, possibly bypassing the transmembrane IFN-alpha/beta receptor. To examine this possibility, cells derived from IFN-alpha/beta Ro/o knockout mice lacking a functional IFN-alpha/beta receptor were stably transformed with pHMB-KbMuIFN beta or pMFG-MuIFN beta plasmids encoding a constitutively expressed murine IFN-beta gene. Four different clones were isolated and examined for resistance to a retrovirus, MFG-LacZ, and to Semliki Forest virus. Despite the production of autocrine IFN-beta at levels inducing high antiviral resistance in control cells, none of the clones displayed antiviral resistance. Thus, despite its failure to be neutralized by potent antiserum, the antiviral activity of autocrine IFN-beta takes place via the transmembrane IFN-alpha/beta receptor, and no additional pathway is involved.

Modifications of CFU-S differentiation pathways by pluripoietins: influence of treatment protocols.

Similar total doses of Ara-C given as a single injection or given in a fractionated protocol have reverse effects on CFU-S differentiation pathways. Whereas a single dose of 20 mg channels CFU-S towards erythropoiesis, 5 X 5 mg given at 8 or 24 hour intervals channels CFU-S to granulopoiesis and megakaryocytopoiesis. It therefore seems possible to manipulate CFU-S differentiation not only by varying the inducing agents but also by varying the protocols using the same agent. Hypotheses to explain the mechanisms of CFU-S regulation are presented.

Further studies on the mechanism of CFU-S determination--I. Pluripoietin(s) responsible for CFU-S determination toward granulopoiesis after fractionated doses of Ara-C.

Fractionated doses of Ara-C orient CFU-S differentiation towards granulopoiesis and megakaryocytopoiesis in vivo in contrast to the preferential channelling towards erythropoiesis after a similar dose of Ara-C given as a single injection. In this paper, we show that pluripoietin found in the serum of mice treated with fractionated doses of Ara-C is responsible for the choice of CFU-S differentiation towards granulopoiesis. Therefore, we confirm our previous results involving single doses of Ara-C, that humoral factors can commit CFU-S preferentially towards one of the cell lineages. It is not as yet known whether there is a specific pluripoietin for each cell lineage or whether the same pluripoietin has a different effect according to its concentration in the organism. The difference in response of CFU-S commitment to single and fractionated doses of Ara-C is not due to the difference in the kinetic status of CFU-S:CFU-S are cycling after both treatments.

Further studies on the mechanism of CFU-S determination--II. Feedback regulation.

In previous papers, we have demonstrated that the determination of CFU-S differentiation is under the control of humoral factors, which we have called pluripoietins. The activity of these regulators varies according to the experimental protocol. The aim of this work was to determine the mechanisms involved in the regulation of pluripoietin secretion after each treatment. The results suggest that the production of pluripoietin(s) is under the control of a feedback mechanism, originating, at least in part, in the progenitor compartments. After one dose of 20 mg of Ara-C, CFU-S determination is channelled towards erythropoiesis when the BFU-E compartment is more depleted than the GM-CFC compartment. During fractionated doses of Ara-C, at the time of the third injection, GM-CFC survival is lower than BFU-E survival and CFU-S orient their differentiation towards granulopoiesis. After bleeding, there is no difference of survival between the two compartments and CFU-S determination is not changed. These results seem to indicate that CFU-S determination is modified in order to restore normal homeostasis of the hemopoietic tissues by preferentially replenishing the more depleted compartment. This is most likely achieved by the secretion of pluripoietin(s) that vary in either their nature or their concentration according to the events occurring in the more mature compartments.

Further studies on the mechanism of CFU-S determination--III. CFU-S cell lineage determination is not influenced by GM-CSF, Epo, multi-CSF nor WEHI 3B conditioned medium.

CFU-S proliferation is under the control of factors such as stimulators and inhibitors whereas commitment of CFU-S is controlled by pluripoietins. We have studied the effects in vitro of Epo, GM-CSF, multi-CSF and WEHI 3B conditioned medium in order to elucidate their eventual role on CFU-S proliferation and cell lineage determination. Neither Epo nor GM-CSF are able to induce the entry of CFU-S into cycle. Multi-CSF and WEHI 3B conditioned medium (similar to interleukin 3) stimulate CFU-S into DNA synthesis. However, we were able to show that the stimulator we have studied is different from IL 3. Epo, GM-CSF, multi-CSF and WEHI 3B CM have no effect on CFU-S commitment. Therefore, these four hemopoietic regulators are different from the pluripoietins we have previously described and have no influence on CFU-S determination.

[Development of an anti-HIV gene therapy based on the antiviral properties of beta interferon]. / Développement d'une génothérapie anti-VIH basée sur les propriétés antivirales de l'interféron bêta.

The aim of our work is to explore the use of IFN-beta for gene therapy in the HIV-infection. Transduction of various HIV target cells with a retroviral vector that carries the Hu-IFN-beta coding sequence under constitutive low expression control, confers resistance to HIV without affecting cell replication or function. After transduction, lymphocytes from HIV-infected patients develop resistance to the endogenous virus, provided the cells are derived from individuals with a CD4 cell count higher than 200 per mm3.

Stable antiviral expression (SAVE) as an approach to somatic cell gene therapy directed against HIV infection.

We are developing methods for somatic cell gene therapy directed against infection with human immunodeficiency virus by enhancing the antiviral resistance of target cells through the constitutive production of interferon-beta. Cells that have been transformed by plasmids or retroviral vectors carrying the human interferon-beta gene placed under the expression control of a murine H2Kb promoter fragment become resistant to HIV infection. Part of this enhanced resistance is due to inhibition of virus entry into the transformed cells, a hitherto unreported mechanism of interferon action.

Inhibition of gene expression at the translational level by cotransfection with competitor plasmids.

We have investigated the effect of cotransfection with other recombinant plasmids on the expression of a test plasmid, p2A9PrCAT, in which the calcyclin promoter directs the expression of the bacterial chloramphenicol transferase (CAT) coding sequence. The expression of CAT is markedly inhibited when p2A9PrCAT is cotransfected with any of several plasmids in which a promoter controls the expression of complete or partial transcribed sequences. A series of experiments indicates that the inhibition is not sequence specific, does not require a full-length protein product, but it requires a transcript, since cotransfection with a vector containing only a promoter gives only a modest inhibition. Although CAT activity is decreased markedly in these competition experiments, the amount of CAT mRNA is unaffected, indicating that the inhibition must take place at a translational level.

Growth factor regulation of the promoter for calcyclin, a growth-regulated gene.

The steady-state levels of calcyclin mRNA are regulated by growth factors. Using deletion mutants of the 5'-flanking region and a linked reporter (the bacterial chloroamphenicol transferase gene), we have investigated the elements of the calcyclin gene's promoter that respond to growth factors. By a transient expression assay after transfection in BALB/c/3T3 cells, we have been able to show that the serum-inducible sequences are contained in a 164-base pair fragment just upstream of the cap site. This fragment also contains an enhancer, and responds to platelet-derived growth factor as well as to serum. The sequences from -1371 to -1194 upstream of the cap site contain an element which is negatively regulated by epidermal growth factor. These findings have been confirmed in hamster cell lines in which the deletion mutants of the calcyclin promoter controlled the expression of the cDNA for human thymidine kinase. These results indicate that, like in other growth-regulated genes the activity of the calcyclin promoter is modulated by both positive and negative elements. Even more intriguing, though, is the finding that some of these negative elements may be influenced by growth factors in the environment.

Blocking of retroviral infection at a step prior to reverse transcription in cells transformed to constitutively express interferon beta.

We are developing methods for somatic-cell gene therapy directed against infection with human immunodeficiency virus, by enhancing antiviral resistance of target cells through the constitutive production of autocrine interferon (IFN). Using the human IFN-beta coding sequence under the constitutive low-expression control of a 0.6-kb murine H-2Kb promoter-fragment, we have constructed a retroviral vector, HMB-KbHuIFN beta, and have transformed cells of the T98G human neuroblastoma line, the U-937 human promonocytic line, and the CEM human lymphocytic line. These human IFN-beta-transformed cell populations have acquired a low, constitutive production of human IFN, while replicating at a rate similar to that of untransformed cells and of cells transformed with the control vector carrying a human IFN-beta sequence encoding an inactive, mutated protein. In the three different cell populations tested, transformation with the HMB-KbHuIFN beta vector resulted in a 1.3-2.3 log10 reduction in the number of cells infected with a defective amphotropic MFG-LaZ retrovirus. A kinetic study of the fate of the MFG-LacZ retrovirus in the culture medium and intracellularly immediately after exposure of the cells to virus revealed a significant reduction of the appearance of intracellular virus in human IFN-beta-transformed cells. A similar effect was obtained by treating untransformed T98G, U-937, and CEM cells with exogenous human IFN-beta. The blocking effect of autocrine or exogenous human IFN-beta on viral entry was not limited to virus specific for the amphotropic receptor but was also obtained in murine IFN-beta-treated NIH 3T3 mouse fibroblasts infected with an ecotropic MFG-LacZ retrovirus. Infection of human IFN-beta-transformed CEM cells with human immunodeficiency virus type 1 gave comparable results. Immediately following exposure of the cells to human immunodeficiency virus, a kinetic study of the fate of the virus failed to reveal the appearance of intracellular virus and showed that the majority of the input virus remained in the extracellular medium. We conclude that low autocrine IFN-beta synthesis, or exposure of cells to exogenous IFN-beta, prevents virus from getting inside the cells, regardless of the virus receptor involved.