The development and characterization of an HEK293-derived cell line for use in an intratumoral cytokine delivery system.

As part of ongoing work to develop a method of cytokine delivery for use as an intratumoral depot, we noted that HEK293 cells, encapsulated in alginate, died within 24-48 h after in vivo, intratumoral implantation. We hypothesized that the highly hypoxic and acidic conditions found inside the tumor was the cause of the cells' premature demise. Therefore, we set out to develop a cell line, derived from HEK293, that would survive these hostile conditions. The HEK293 line was selected in 0.3-0.5% oxygen conditions over several weeks, followed by a further 6-week period of culture in alternating hypoxic and normoxic conditions. The most rapidly growing clones were selected and grown in normoxic conditions for several weeks to ensure their stability. The clones were then compared to the original line in terms of cell proliferation in normoxia and hypoxia, colony-forming efficiency, and morphological characteristics. The resulting line was able to proliferate in the harshest of conditions and continues to release its biological payload after alginate microencapsulation.

The Development and Characterization of an HEK293-Derived Cell Line for Use in an Intratumoral Cytokine Delivery System.

As part of ongoing work to develop a method of cytokine delivery for use as an intratumoral depot, we noted that HEK293 cells, encapsulated in alginate, died within 24-48 h after in vivo, intratumoral implantation. We hypothesized that the highly hypoxic and acidic conditions found inside the tumor was the cause of the cells' premature demise. Therefore, we set out to develop a cell line, derived from HEK293, that would survive these hostile conditions. The HEK293 line was selected in 0.3-0.5% oxygen conditions over several weeks, followed by a further 6-week period of culture in alternating hypoxic and normoxic conditions. The most rapidly growing clones were selected and grown in normoxic conditions for several weeks to ensure their stability. The clones were then compared to the original line in terms of cell proliferation in normoxia and hypoxia, colony-forming efficiency, and morphological characteristics. The resulting line was able to proliferate in the harshest of conditions and continues to release its biological payload after alginate microencapsulation.

VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death.

BACKGROUND: The inefficiency of herpes simplex virus thymidine kinase (TK) gene transfer and toxicity of ganciclovir (GCV) at high concentrations in vivo limits the use of this suicide gene therapy approach for the treatment of cancers in clinical settings. To overcome the problem, we have sought evidence of amplification of cytotoxicity by co-transfer of the TK gene fused with the gene encoding HSV-1 structural protein VP22 which has a remarkable ability for intercellular trafficking. METHODS: The expression of the fusion proteins from the chimeric VP22-TK or VP22-EGFP genes was shown by Western blot and VP22 promoted TK or EGFP intercellular trafficking by an indirect immunofluorescent assay. The cytotoxicity was demonstrated by a colorimetric cell proliferation assay followed by an assessment of the bystander effect on admixtures of transfected with non-transfected naive cells. RESULTS: Our results show the expression of the VP22 fusion proteins and their spread to varying numbers of bystander cells (up to 30, observed in viable cells with VP22-EGFP as well as after methanol fixation), confirming that VP22 assisted intercellular trafficking of the fusion proteins. This VP22 promoted TK spreading resulted in killing by 2.5 microg/ml GCV of virtually all cells in cultures that had been transfected at an efficiency of only 27.5%. In contrast, fewer than 80% of cells were killed when transfected with 'tk alone' at the same efficiency. The cell killing effect was exponentially dependent on GCV concentration in cells transfected with 'tk alone' at GCV concentrations between 0.25 and 0.5 microg/ml, but not those transfected with VP22-TK, probably due to the continuously variable, high sensitivity of about 50% of cells. Even at low concentration of GCV (0.2 microg/ml), the enhancement of cell killing by VP22 was four-fold higher in cells transfected with VP22-TK than in cells transfected with 'tk alone'. CONCLUSIONS: VP22 enhanced intercellular trafficking of TK and amplified the TK/GCV killing effect, especially in the lower range of GCV concentrations. This offers a new strategy to enhance the effectiveness of suicide gene therapy for the treatment of cancers.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle.

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.

Lentiviral vector-mediated tyrosinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR' CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4+ and CD8+ cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Increased cell surface protease activity in UV-irradiated cells undergoing apoptosis.

UVB-irradiated HeLa cells undergoing apoptosis have increased cell surface protease (CSP) activity compared to viable or necrotic cells. In order to elucidate whether caspase 3 plays a role in the activation of CSP in cells undergoing apoptosis, HeLa cell cultures were pre-treated with the caspase inhibitor, DEVD, prior to being exposed to 500 Jm(-2) UVB. DEVD significantly inhibited caspase 3 activity in cells undergoing apoptosis, but did not affect the activation of CSP in these cells. The findings suggest that the activation of CSP in apoptotic cells is unrelated to caspase 3 activity.

Novel bovine lentiviral vectors based on Jembrana disease virus.

BACKGROUND: Safety is a concern that must be addressed prior to any clinical use of human immunodeficiency virus (HIV)-based lentiviral vectors in human patients. Unfortunately, efforts to examine the biosafety of the vectors in preclinical animal models are hampered due to the lack of animal models for HIV infection. We have developed new lentiviral vectors based on the recently characterised Jembrana Disease Virus (JDV), which infects a specific species of cattle naturally in Bali, Indonesia. METHODS: Sequences from the JDV genome were amplified by splicing overlap extension polymerase chain reaction (PCR) for the construction of transfer vectors as well as a packaging construct. Co-transfection of these two plasmids into 293T cells with a third encoding a G glycoprotein of vesicular stomatitis virus produced pseudotyped, disabled, replication defective JDV vector particles. Viral titre was obtained by transducing the cells with the supernatant harvested from transfectants and determining the number of cells expressing the transgene. PCR and Southern blotting were used to detect the presence of potential replication-competent viruses as well as transgene integration. RESULTS: Bicistronic JDV vectors encoding the green fluorescent protein (GFP) and the neomycin phosphotransferase were harvested with a titre range of 0.4-1.2 x 10(6) colony forming units/ml from vector-producing cells and were further concentrated by ultracentrifugation to the high titre of approximately 10(7) CFU/ml. Vectors encoding GFP were shown to transduce and integrate efficiently into the chromosomes of a range of primary and transformed cells of different origins in different differentiation status, including growth-arrested cells, with an efficiency of 25-75%. Exhaustive testing with a marker gene transfer assay in combination with a reverse transcriptase assay and PCR amplification of samples of serially passaged, transduced cells showed that no detectable amount of replication competent lentivirus (RCL) was produced. CONCLUSIONS: We showed the feasibility of the development of gene transfer vectors based on a non-primate bovine lentivirus, which will provide the opportunity for examination of the efficacy and biosafety of lentiviral vector-mediated gene transfer in vivo in animal models. JDV-based vectors may be applicable and more readily acceptable than those from HIV for human gene therapy.

Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events.

The activation of cdc2/cyclin B is the trigger for entry into mitosis. The mechanism of cdc2/cyclin B activation is complex, but the final step is the dephosphorylation of the Thr14 and Tyr15 residues on the cdc2 subunit, catalyzed by a member of the Cdc25 family of phosphatases. Cdc2/cyclin B1 accumulates at the centrosome in late G2 phase and has been implicated in the conversion of the centrosome from an interphase to a mitotic microtubule organizing center. Here we demonstrate biochemically that cdc2/cyclin B1 accumulates at the centrosome in late G2 as the inactive, phosphotyrosine 15 form and that the centrosomal cdc2/cyclin B1 can be activated in vitro by recombinant cdc25B. We provide evidence that a portion of the cdc2/cyclin B1 translocated into the nucleus in prophase is the inactive tyrosine-15-phosphorylated form. At this time the centrosomal and cytoplasmic cdc2/cyclin B1 is already active. This provides evidence that the activation of cdc2/cyclin B1 is initiated in the cytoplasm and that full activation of the translocated pool occurs in the nucleus.

Increased ecto-metallopeptidase activity in cells undergoing apoptosis.

Release from the cell surface of a variety of growth factors, cytokines, and proteases follows exposure to genetically stressful agents capable of inducing apoptosis and necrosis. Increased ectoprotease activity is responsible for their release. We show that increased activity of several metalloproteases on the HeLa cell surface occurs after stresses due to UVC, actinomycin D, cycloheximide, and cisplatinum, which induce the release of transforming growth factor-alpha (TGFalpha) and other bioactive molecules. The ectoprotease activities increase preferentially on apoptotic cells, while little change occurs in viable cells. Gross decreases, except for the putative TGFalphaase activity, accompany necrosis. These changes may contribute to tissue repair and the absence of an inflammatory reaction to apoptotic cell death. They appear to be due to preferential enzyme activation or to retention by cells undergoing significant categorical decreases in protein content.

John Kerr and apoptosis.

On 14 March 2000, John Foxton Ross Kerr, Emeritus Professor of Pathology at the University of Queensland, received the Paul Ehrlich and Ludwig Darmstaedter Prize for his description of apoptosis, a form of cell death. The prize, which he shared with Boston biologist Robert Horvitz, is considered to be one of the most prestigious European awards in science, second only to the Nobel Prize.

Multiple splicing variants of cdc25B regulate G2/M progression.

Progression through G2 phase into mitosis is regulated by the activation of the mitotic cyclin/cdk complexes, which are in turn activated cdc25B and cdc25C phosphatases. Here we report that alternate splicing produces at least five variants of cdc25B, although only cdc25B2 and cdc25B3 are detectable as proteins. Analysis of these two variants shows that cdc25B2 is expressed at lower levels relative to cdc25B3 in all cell lines tested, and the expression of both increased markedly during G2 and mitosis. Overexpression of the catalytically inactive version of either cdc25B variant produced a G2 arrest implicating both in regulating G2/M progression.

A cyclin D-Cdk4 activity required for G2 phase cell cycle progression is inhibited in ultraviolet radiation-induced G2 phase delay.

Cyclin D-Cdk4 complexes have a demonstrated role in G1 phase, regulating the function of the retinoblastoma susceptibility gene product (Rb). Previously, we have shown that following treatment with low doses of UV radiation, cell lines that express wild-type p16 and Cdk4 responded with a G2 phase cell cycle delay. The UV-responsive lines contained elevated levels of p16 post-treatment, and the accumulation of p16 correlated with the G2 delay. Here we report that in UV-irradiated HeLa and A2058 cells, p16 bound Cdk4 and Cdk6 complexes with increased avidity and inhibited a cyclin D3-Cdk4 complex normally activated in late S/early G2 phase. Activation of this complex was correlated with the caffeine-induced release from the UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of Cdk4 blocked cells in G2 phase. These data indicate that the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 phase into mitosis and that the increased binding of p16 blocks this activity and G2 phase progression after UV exposure.

Involvement of p16CDKN2A in cell cycle delays after low dose UV irradiation.

Ultraviolet (UV) radiation contributes to the aetiology of melanoma, but the precise mechanistic details are still unclear. The CDKN2A gene which is associated with familial and sporadic melanoma, encodes a tumour suppressor, p16. We have previously shown that in response to low doses of UV radiation the level of p16 increases, and that this correlates with a G2 delay. Here we report that in melanoma cell lines which do not express p16, or express a mutant p16, no G2 delay is observed in response to UV. The loss of functional p16 also correlates with an increase in DNA damage as judged by increased numbers of bi- and multinuclear cells and cells containing 1-2 micronuclei following UV irradiation. This work provides a further link between UV radiation, CDKN2A and melanoma, suggesting that the functional inactivation of CDKN2A disrupts a p16-dependent G2 cell cycle checkpoint, thus contributing to the development of this neoplasm.

Effect of UV irradiation on cell surface protease activity and amino acid uptake.

The surface of most cells includes a coterie of resident proteins which act as receptors for a wide variety of ligands and other proteins which are potentially bioactive on cell-cell contact (juxtacrine effects), or else are released by enzyme activity to influence cell behaviour by autocrine or paracrine mechanisms. We previously found that UVC irradiation stimulates the release of TGFalpha from its membrane-bound preprocursor form whereby it acts as a stimulus to rapid, reparative cell multiplication; clearly this runs the risk hastening mitosis before UV-induced DNA damage is fully corrected, which in turn may increase the likelihood of residual lesions persisting and hence of new mutations being generated. We found that sublethal UVC irradiation (10 J m(-2)) of HeLa cell cultures also resulted in activation of ecto-aminopeptidase and ecto-endopeptidases which were maximal 16 and 20-24 h after irradiation, respectively. Both of these classes of protease were shown to be metalloproteases using a nonapeptide substrate (called P9) which is cognate to the N-terminal cleavage site of preproTGFalpha except for a reporter 125I-tyrosine [Piva et al., J. Cell. Biochem. 64 (1997) 353-368]. We now show that the N-terminal tyrosine cleaved from P9 by cell surface aminopeptidase activity, was found to be taken up by the cell resulting in its 10-25-fold concentration intracellularly, some two- to threefold higher than from a reservoir source, and may represent a novel salvage pathway for recovery of essential amino acids. Aminopeptidase activity was found to be both temperature- and FBS-dependent but was not reliant on ATP for its activity. Tyrosine transport across the cell membrane was also temperature and FBS-dependent but required ATP for maximal activity. UVC irradiation enhanced aminopeptidase activity but not tyrosine uptake by the cultures. The fraction of HeLa cells undergoing apoptosis increased in those cultures which were exposed to higher doses of UVC. The levels of ecto-aminopeptidase and ecto-endopeptidase activity in apoptotic cells were elevated compared to viable cells receiving the same dose of UVC. These results suggest that increased levels of cell surface protease activity in apoptotic cells would increase the amounts of free amino acids and growth factors in the extracellular medium and hence stimulate the proliferation of surrounding cells to replace those killed by UV irradiation.

Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2.

Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G2/M transition. Here we show, using bacterially expressed recombinant proteins, that two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form, and following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This was reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of cdc25B and cdc25C during G2 and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G2 into mitosis.

Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical adenocarcinoma cell line HeLa.

In this study we describe the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein.

Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis.

In response to low doses of ultraviolet (U.V.) radiation, cells undergo a G2 delay. In this study we have shown that the G2 delay results in the accumulation of inactive forms of cyclin B1/cdc2 and both the G2 and mitotic complexes of cyclin A/cdk. This appears to be through a block in the cdc25-dependent activation of these complexes. The expression and localisation of cyclin A and cyclin B1/cdk complexes are similar in U.V.-induced G2 delay and normal early G2 phase cells. Cdc25B and cdc25C also accumulate to normal G2 levels in U.V. irradiated cells, but the mitotic phosphorylation associated with increased activity of both cdc25B and cdc25C is absent. The cdc25B accumulates in the nucleus of U.V. irradiated cells and in normal G2 phase cells. Thus the block in cyclin B/cdc2 activation is in part due to the physical separation of cyclin B/cdc2, localised in the cytoplasm, from the cdc25B and cdc25C phosphatases localised in the nucleus. The data positions the U.V.-induced G2 checkpoint at either the S/G2 transition or early G2 phase, prior to the activation of cyclin A/cdk2.

A case report: immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy.

The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient, with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8+ response to a high proportion of CD4+ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophila. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases: however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient.

Immunotherapy, including gene therapy, for metastatic melanoma.

Current standard therapy for distant metastatic melanoma is ineffective and often compromises the quality of a patient's life. Immunotherapy is briefly reviewed in relation to its many forms: from local non-specific to the more recent specific vaccines, including those using specific melanoma peptides (e.g. from the proteins encoded by melanoma-associated gene (MAGE)) and those involving genetically transduced autologous melanoma cells using retroviral vectors in vitro. The mode of action of genetically transduced melanoma cells incorporating the granulocyte macrophage colony stimulating factor (GM-CSF) gene (GVAX) is presented as a paradigm for cytokine-mediated strategies. Trials of GVAX and other cytokine gene strategies are under way in Brisbane, Boston and Amsterdam, and some interim perspectives on the clinical outcomes and immunological mechanisms involved are sketched. Some of the compounding problems in immunotherapeutic strategies for cancer are identified, and possible adjunct manoeuvres for overcoming them are discussed.