PG13 packaging cells produce recombinant retroviruses carrying a diphtheria toxin mutant which kills cancer cells

The development of suicide gene therapy with gene products that are directly toxic to cells, such as the A subunit of diphtheria toxin (DT-A), has been hampered by the difficulty of engineering recombinant viruses. DT-A is a strong inhibitor of protein synthesis that acts by ADP-ribosylating elongation factor 2, and a low level of DT-A expression in virus producer cells prevents the production of recombinant virus. We analyzed here the natural resistance of packaging cells to DT-A toxicity, and we report that PG13 and PA317 packaging cell lines are resistant to H21G, a DT-A mutant. PG13 cells produce recombinant H21G virus that efficiently kills a variety of human tumor cells. Our finding indicates that PG13 packaging cells provide a new potential for the development of DT-A-based suicide gene therapy.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

Use of recombinant retroviruses to characterize the activity of antiretroviral compounds.

This report describes the use of a recombinant murine retrovirus encoding beta-galactosidase (PLJ beta-gal retrovirus) to study the antiretroviral activity of zidovudine (AZT) and other nucleoside analogs. The PLJ beta-gal virus permits the rapid and unequivocal identification of individual virus-infected cells arising from a single cycle of viral replication. With this model system, AZT is shown to completely and irreversibly prevent retrovirus infection of proliferating cell lines as measured by a lack of reporter gene expression. On the other hand, AZT is less effective in protecting growth-arrested cells from retroviral infection. Recombinant retroviruses such as the PLJ beta-gal virus are potentially useful reagents for the identification and characterization of antiretroviral compounds.     

Summary of recombinant human serum albumin development.

The methylotrophic yeast Pichia pastoris is well known as a host strain for the production of a variety of heterologous proteins. We have used P. pastoris for the production of recombinant human serum albumin (rHSA), for which we have developed efficient and specialized downstream processes. Results from structural analysis suggest that purified rHSA possesses an identical conformation to plasma derived human albumin (pdHA) and no difference from pdHA has been observed in neo-antigenicity. Host-cell-derived impurities (i.e. Pichia yeast component, DNA and mannan) have been evaluated in the purification process as well as in the drug substance and relevant specifications established. The efficacy and safety of rHSA have been tested in clinical studies and no difference from pdHA has been found in comparative study. Such studies have confirmed rHSA to have high efficacy with little or no adverse reaction.     

High-titer replication of nondefective Sendai virus in MDBK cells.

Egg-grown Sendai virus was adapted to growth in a bovine kidney cell line (MDBK cells) by serial passage under defined conditions. The adapted virus contained only 50S RNA and was highly infectious for MDBK cells. Infection of these cells with a high multiplicity of adapted virus resulted in a yield of 10(8) MDBK-infectious units/ml by 18 h, accompanied by severe cytopathic changes in the host. Cell fusion did not occur. Examination of the proteins of the adapted virus revealed that despite the high infectivity of this virus for MDBK cells the virions contained considerable quantities of Fo, the precursor to the F glycoprotein that is responsible for cell fusion and high infectivity in other systems.     

Introduction of human genomic sequences renders CHO-K1 cells susceptible to infection by amphotropic retroviruses.

To learn more about the nature of the block to infection by amphotropic retroviruses exhibited by Chinese hamster cells (CHO-K1), CHO-K1 cells were made susceptible to amphotropic retrovirus infection by introducing genomic DNA from infectable human cells. A clone, designated CHO18, was obtained and shown to be infected as efficiently as NIH 3T3 fibroblasts. Susceptibility of CHO18 cells to infection was specific to retroviruses and vectors bearing an amphotropic envelope. By comparison to CHO-K1 cells, CHO18 cells may provide a useful model for analysis of the molecular events involved in the retrovirus-receptor interaction.     

Use of recombinant retroviruses to study the regulation of integrated adenovirus early promoters.

Adenovirus E1A gene products are capable of modulating the expression of a variety of integrated genes. To study the mechanisms by which this regulation occurs, recombinant retroviruses have been utilized to establish cell lines containing an integrated copy of either the adenovirus E2 or E3 promoter adjacent to the bacterial guanine phosphoribosyl transferase (GPT) gene. These cell lines have been characterized with respect to both basal and E1A-induced levels of GPT gene expression. Cell lines with low levels of GPT gene expression showed increased expression in the presence of E1A, whereas cell lines with high basal levels of GPT gene expression had decreased GPT RNA levels in the presence of E1A. Further characterization of these cell lines revealed E1A modulation of the accumulation of RNA initiating at a retrovirus promoter adjacent to the E2 or E3 promoter. The use of the GPT gene as a marker of E2 or E3 promoter activity has allowed the isolation of cell lines which have spontaneously increased their levels of GPT RNA. A preliminary characterization of four of these cell lines has indicated that GPT gene expression is increased as a result of cis activation of the E2 promoter.     

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.     

Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus.

The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.     

Detection of recombinant P-glycoprotein in multidrug resistant cultured cells

The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells. Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer. Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers. Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents. P-glycoprotein was found to be overexpressed in many of these models. Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells. Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions. Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes. This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells. Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein. Strategies to visualize P-glycoprotein include metabolic labeling using ³⁵S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.     

Multiplex genome editing of mammalian cells for producing recombinant heparin

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.     

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.     

Human recombinant migration inhibitory factor activates human macrophages to kill tumor cells.

A recombinant form of human migration inhibitory factor (rMIF) obtained from COS-1 cells transfected with MIF-specific cDNA is able to activate cultured human peripheral blood monocytes and monocyte-derived macrophages, in a dose-dependent manner to become cytotoxic for tumor cells in vitro. The cytotoxicity exhibited by macrophages treated with rMIF is > or = 30% above that of cells incubated with control supernatants or with media and peaks 72 hr after the addition of tumor targets. rMIF also induces macrophages to produce tumor necrosis factor (TNF-alpha) and interleukin-1 beta (IL-1 beta). These results demonstrate that rMIF is able to modulate macrophage functions and plays a role in cell-mediated immune response.     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.