Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells.

Dendritic cells (DCs) are potent antigen-presenting cells that prime naive cytotoxic T-cells (CTLs). In this study, we have fused DCs with MC38 carcinoma cells. The fusion cells were positive for major histocompatibility (MHC) class I and II, costimulating molecules and intercellular cell adhesion molecule-1 (ICAM-1). The results show that the fusion cells stimulate naive T cells in the primary mixed lymphocyte reaction (MLR) and induce MC38 tumor-specific CTLs in vivo. Antibody-mediated depletion experiments demonstrate that induction of CD4+ and CD8+ CTLs protects against challenge with tumor cells. We also show that immunization with the fusion cells induces rejection of established metastases. These findings represent the first demonstration that fusions of DCs and tumor cells can be used in the treatment of cancer.

Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth.

Chronic systemic delivery of therapeutic proteins, such as inhibitors of angiogenesis, present a number of difficult pharmacological challenges. To overcome these problems for one such protein, we constructed retroviral and adenoviral vectors that express a novel, secretable form of the antiangiogenic protein, platelet factor 4 (sPF4). Vector-mediated sPF4 transduction selectively inhibits endothelial cell proliferation in vitro, and results in hypovascular tumors that grow slowly in vivo. Additionally, tumor-associated angiogenesis is inhibited and animal survival is prolonged, following transduction of established intracerebral gliomas by an sPF4-expressing adenoviral vector. These data support the concept that targeted antiangiogenesis, using virally mediated gene transfer, represents a promising strategy for delivering antiangiogenic therapy.

Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo.

Cytosine arabinoside (ara-C) is a cytidine analog that incorporates into replicating DNA and induces lethal DNA strand breaks. Although ara-C is a potent antitumor agent for hematologic malignancies, it has only minimal activity against most solid tumors. The rate-limiting step in intracellular ara-C activation is phosphorylation of the prodrug by deoxycytidine kinase (dCK). The present results demonstrate that both retroviral and adenoviral vector-mediated transduction of the dCK cDNA results in marked sensitization of glioma cells lines to the cytotoxic effects of ara-C in vitro. We also demonstrate that ara-C treatment of established intradermal and intracerebral gliomas transduced with dCK results in significant antitumor effects in vivo. These data suggest that viral vector transduction of the dCK gene followed by treatment with ara-C represents a new chemosensitization strategy for cancer gene therapy.

Tumor-selective transgene expression in vivo mediated by an E2F-responsive adenoviral vector.

Recent data suggest that many tumors, such as malignant gliomas, have disrupted pRB function, either because of RB-1 gene mutations or as a result of mutations affecting upstream regulators of pRB such as cyclin D1 or p16/INK4a/MTS1 (ref. 1-5). Tumor suppression by pRB has been linked to its ability to repress E2F-responsive promoters such as the E2F-1 promoter. Thus, a prediction, which has not yet been demonstrated experimentally in vivo, is that E2F-responsive promoters should be more active in tumor cells relative to normal cells because of an excess of "free" E2F and loss of pRB/E2F repressor complexes. We demonstrate that adenoviral vectors that contain transgenes driven by the E2F-1 promoter can mediate tumor-selective gene expression in vivo, allowing for eradication of established gliomas with significantly less normal tissue toxicity than seen with standard adenoviral vectors. Our data indicate that de-repression of the E2F-1 promoter occurs in cancer cells in vivo, a finding that can be exploited to design viral vectors that mediate tumor-selective gene expression.

Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.

Given the plethora of well-documented breast carcinoma-associated antigens in humans including MAGE-1, -2 and -3, mutated p53, p21ras, HER-2/neu and DF3/MUC-1, coupled with evidence that humoral and cytotoxic T-cell responses against these antigens exist, the central dilemma facing tumor immunologists is why the host immune response is so inefficient. One possibility is that tumor cells themselves are either inefficient or ineffective antigen-presenting cells (APCs). The failure of tumor cells to function as APCs may be due to their inability to process and present the antigen, the absence or insufficient numbers of adhesion and costimulatory molecules or, potentially, the secretion of inhibitory cytokines. Therefore, we sought to determine whether human breast cancer cell lines could function as APCs and, if not, to identify mechanism(s) responsible for this defect. Here, we show that human breast cancer cell lines fail to present alloantigen. This defect does not reside in their inherent capacity to present antigen but rather is due to apoptosis of activated T cells induced by exposure to the breast carcinoma-associated mucin antigen, DF3/MUC1. These results support the hypothesis that DF3/MUC1 may contribute to the paucity of clinically significant anticarcinoma-specific immune responses.

Spatial and temporal control of gene therapy using ionizing radiation.

Activation of transcription of the Egr-1 gene by X-rays is regulated by the promoter region of this gene. We linked the radiation-inducible promoter region of the Egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (TNF-alpha) and used a replication-deficient adenovirus to deliver the Egr-TNF construct to human tumours growing in nude mice. Combined treatment with Ad5.Egr-TNF and 5,000 cGy (rad) resulted in increased intratumoral TNF-alpha production and increased tumour control compared with treatment with Ad5.Egr-TNF alone or with radiation alone. The increase in tumour control was achieved without an increase in normal tissue damage when compared to tissue injury from radiation alone. Control of gene transcription by ionizing radiation in vivo represents a novel method of spatial and temporal regulation of gene-based medical treatments.

Circulating interleukin 6 during a continuous infusion of tumor necrosis factor and interferon gamma.

Plasma samples obtained from patients receiving a 24-h continuous infusion of human recombinant (hr)TNF or a combination of two overlapping, 24-h continuous infusions of hrIFN-gamma and hrTNF were analyzed for IL-6 in a sensitive bioassay. A transient appearance of circulating IL-6 was observed with peak levels between 3 and 6 h after the start of the hrTNF infusion. These peak levels correlated quite well with the dose of hrTNF administered (r = 0.86; p less than 0.001). The maximal value observed was 27.5 ng/ml IL-6 in a sample of a patient receiving 545 micrograms/m2 hrTNF. The combination of hrIFN-gamma (200 micrograms/m2) and hrTNF in the infusions resulted in higher IL-6 levels than a comparable dose of hrTNF alone. A maximal value of 23.5 ng/ml IL-6 was observed in a patient receiving 205 micrograms/m2 hrTNF. No IL-6 was found in the plasma of patients during the 12-h infusion with hrIFN-gamma alone, except for two borderline samples.

Oncostatin M induces association of Grb2 with Janus kinase JAK2 in multiple myeloma cells.

Oncostatin M (OSM) is a 28-kD glycoprotein recently identified as a growth factor for human multiple myeloma cells. It belongs to a family of distantly related cytokines that includes interleukin 6, ciliary neurotrophic factor, leukemia-inhibitory factor, and interleukin 11. These cytokines initiate signaling by inducing either homodimerization of gp130 or heterodimerization of gp130 with leukemia-inhibitory factor receptor beta components. Such dimerization in turn activates receptor-associated tyrosine kinases. In the present study using U266B1 human multiple myeloma cells, we show that OSM induces tyrosine phosphorylation and activation of JAK2, but not JAK1 or Tyk2, kinases. The results also demonstrate that OSM induces direct interaction of JAK2 kinase with Grb2, an SH2/SH3 domain containing adaptor protein. The SH2 domain of Grb2 is directly associated with tyrosine-phosphorylated JAK2. Furthermore, the presence of Sos in the JAK2-Grb2 complex suggests a role for Ras in OSM-transduced signaling.

Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

Human sarcomas contain RNA related to the RNA of a mouse leukemia virus.

Labeled DNA complementary to the RNA of the Rauscher leukemia virus was hybridized with RNA from the polysome fraction of human sarcomas. Eighteen out of 25 specimens contained RNA possessing homology to the RNA of the mouse leukemia virus but not to that of the unrelated viruses causing mammary tumors in mice or myeloblastosis in chickens. Further, no normal adult or fetal tissues showed significant amounts of RNA specific to mouse leukemia virus. It appears that human sarcomas contain RNA sequences homologous to those found in an agent related to a virus known to cause sarcomas in mice.

MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90.

The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit localizes to mitochondria and blocks stress-induced activation of the intrinsic apoptotic pathway. How MUC1 is delivered to mitochondria is not known. The present studies demonstrate that MUC1 forms intracellular complexes with HSP70 and HSP90. We show that the MUC1 cytoplasmic domain binds directly to HSP70 in vitro. By contrast, binding of MUC1 to HSP90 in vitro is induced by c-Src-mediated phosphorylation of the MUC1 cytoplasmic domain. c-Src also increases binding of MUC1 to HSP90 in cells. In concert with these results, we show that heregulin (HRG), a ligand for ErbB receptors, activates c-Src and, in turn, stimulates binding of MUC1 to HSP90. We also show that inhibitors of c-Src or HSP90 block HRG-induced targeting of MUC1 to mitochondria and integration of MUC1 into the mitochondrial outer membrane. These findings indicate that MUC1 is delivered to mitochondria by a mechanism involving activation of the ErbB receptor-->c-Src pathway and transport by the molecular chaperone HSP70/HSP90 complex.

Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase.

BACKGROUND: Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. RESULTS: We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. CONCLUSIONS: The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

Internucleosomal DNA fragmentation during phorbol ester-induced monocytic differentiation and G0/G1 arrest.

The treatment of human myeloid leukemia cell lines with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), is associated with loss of proliferative capacity and induction of monocytic differentiation. The present results demonstrate that treatment of asynchronous human U-937 leukemia cells with 10 nM TPA is also associated with oligonucleosomal DNA cleavage. This pattern of DNA fragmentation, which is observed in programmed cell death, was detectable in populations of TPA-treated cells that had entered a nonproliferative G0/G1 phase. Similar findings were obtained after TPA treatment of a synchronous population of G1 cells. These cells progressed through S and G2/M phases before undergoing internucleosomal DNA cleavage during G0/G1 arrest. These G0/G1 cells displayed characteristics of monocytic differentiation, including down-regulation of c-myc expression and induction of c-fms transcripts. DNA fragmentation was also studied in cells treated with 5 nM TPA for 48 h and then monitored in drug-free long-term culture. Endonucleolytic cleavage was similarly observed in the differentiated G0/G1 population. However, longer periods of culture were associated with a decrease in DNA fragmentation to undetectable levels. This effect was followed by retrodifferentiation and reentry of cells into cycle. Taken together, these findings demonstrate that internucleosomal DNA fragmentation occurs during induction of monocytic differentiation, and that both of these events are detectable in G0/G1 cells.

Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes.

Regulation of tumor necrosis factor (TNF) gene expression was investigated in resting human monocytes and in 12-O-tetradecanoylphorbol-13-acetate (TPA) activated monocytes. TNF transcripts were undetectable in resting monocytes. However, in TPA-activated monocytes, TNF mRNA was first detectable by 3 h and reached maximal levels by 12 h of drug exposure. Using run-on transcription assays, the TNF gene was transcriptionally inactive in resting monocytes, but was rapidly activated after TPA exposure. The protein synthesis inhibitor, cycloheximide (CHX), had no detectable effect on levels of TNF transcripts in resting monocytes, while this agent superinduced the level of TNF mRNA by 50-fold in TPA-activated cells. TPA activated monocytes were also exposed to actinomycin D and/or CHX to determine whether transcriptional or posttranscriptional control of TNF gene expression was responsible for the induction of TNF transcripts. After 1 h of actinomycin D treatment, the amount of TNF transcripts was reduced by 75%. In contrast, no difference in TNF mRNA levels was observed in TPA-activated monocytes exposed to CHX alone or CHX in combination with actinomycin D. These findings indicated that CHX prevented the degradation of TNF mRNA by inhibiting the synthesis of a labile protein. Run-on transcription assays performed on cells exposed to either TPA or the combination of TPA and CHX further indicated that CHX treatment increased transcription of the TNF gene. Thus, TNF gene expression is controlled at the transcriptional level in resting human monocytes, while both transcriptional and posttranscriptional events regulate the level of TNF transcripts in TPA-activated cells.

Transcriptional regulation of c-jun gene expression by arabinofuranosylcytosine in human myeloid leukemia cells.

Previous studies have demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces terminal differentiation of human myeloid leukemia cells. Other studies have shown that the c-jun protooncogene is expressed during phorbol ester-induced myeloid differentiation. This work examines the effects of ara-C on c-jun gene expression in human KG-1 myeloid leukemia cells. The results demonstrate that c-jun transcripts are undetectable in uninduced KG-1 cells and that ara-C induces expression of this gene in a concentration- and time-dependent manner. Ara-C treatment was also associated with increases in c-jun transcripts in U-937, THP-1, and HL-60 myeloid leukemia cells. Furthermore, transcriptional run-on analysis has demonstrated that exposure to ara-C increases the rate of c-jun gene transcription. The results also demonstrate that while inhibition of protein synthesis superinduces c-jun mRNA levels in phorbol ester-treated KG-1 cells, cycloheximide had no effect on the induction of c-jun transcripts during ara-C treatment. Moreover, the half-life of c-jun transcripts in ara-C-treated KG-1 cells was 42 min. These findings suggest that the increase in c-jun mRNA observed during ara-C treatment is regulated by a transcriptional mechanism, and that c-jun may be involved in the induction of differentiation and regulation of gene expression by ara-C.

Induction of terminal differentiation in human K562 erythroleukemia cells by arabinofuranosylcytosine.

We have previously demonstrated that continuous exposure of human HL-60 human promyelocytes to 1-beta-D-arabinofuranosylcytosine (ara-C) results in the induction of terminal differentiation to monocyte-like cells. The present study extends these findings by demonstrating that ara-C induces hemoglobin synthesis in human K562 erythroleukemia cells. This effect occurs maximally at an ara-C concentration (5 X 10(-7) M) that results in K562 cytostasis. In contrast to the reversible effects of hemin and hydroxyurea on globin synthesis in this cell line, we have found that the induction of K562 hemoglobin synthesis by ara-C is irreversible. An induction of K562 hemoglobin synthesis also occurs with aphidicolin, another inhibitor of S-phase DNA synthesis, but not with vinblastine, an inhibitor of mitosis. Finally, ara-C induction of a differentiated K562 phenotype is accompanied by the loss of self-renewal capacity, a finding consistent with terminal differentiation.

Tumor necrosis factor expression in human epithelial tumor cell lines.

Tumor necrosis factor (TNF) is a monokine with in vitro cytotoxicity for some but not all tumor cells. The basis for sensitivity and resistance to the antitumor effects of this agent remains unclear. The present studies have monitored the effects of TNF on 14 epithelial tumor cell lines. Eleven of these cell lines were resistant to the growth inhibitory effects of TNF (50% inhibitory concentration greater than 1,000 U/ml). 12 of the 14 tumor cell lines has detectable levels of high affinity cell surface TNF binding sites, thus suggesting that resistance was not often due to the absence of cell surface TNF receptors. Northern blot analysis demonstrated that three of the eleven resistant cell lines expressed detectable levels of TNF mRNA. Furthermore, both sensitive and resistant epithelial tumor cells had the capacity to express TNF transcripts in the presence of the protein synthesis inhibitor, cycloheximide. Finally, the presence of TNF expression at the RNA level is shown to be associated with the production of a TNF-like protein in the resistant Ov-D ovarian carcinoma cells. These findings suggest that certain human epithelial tumor cell lines inherently resistant to TNF also express this cytokine.

Expression of platelet-derived growth factor (PDGF)-related transcripts and synthesis of biologically active PDGF-like proteins by human malignant epithelial cell lines.

Human malignant epithelial cell lines were analyzed for expression of platelet-derived growth factor (PDGF) genes. Of the 12 cell lines tested, 9, derived from breast, lung, gastric, and ovarian carcinomas, were found to express both PDGF-1 and PDGF-2 genes. The levels of both PDGF-1 and PDGF-2 transcripts were superinduced when these cells were treated with cycloheximide, an inhibitor of protein synthesis. These cells also released an activity that in studies with BALB-c/3T3 cells, inhibited binding of 125I-labeled PDGF and stimulated incorporation of [3H]thymidine. This stimulating activity was inhibited after reduction of the conditioned media by mercaptoethanol or after preincubation with antibodies to PDGF. Moreover, this activity was not affected by heat treatment. Immunoprecipitation studies revealed that breast, lung, and gastric carcinoma cells produced PDGF-like proteins that migrated as 30- and 32-kD species under nonreducing conditions and as 15- and 16-kD species under reducing conditions. In contrast, malignant cells of ovarian origin produced 14-16-kD PDGF-like proteins that were unchanged in mobility after reduction. As PDGF receptors were not detected on these malignant epithelial cells, the production of PDGF-like proteins may affect other cells in the microenvironment by paracrine mechanisms and may contribute to excessive cell proliferation, inflammatory reactions, and connective tissue remodeling seen in certain carcinomas.

Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen.

The DF3/MUC1 gene is aberrantly overexpressed in human breast and other carcinomas. Previous studies have demonstrated that the DF3/MUC1 promoter/enhancer confers selective expression of diverse transgenes in MUC1-positive breast cancer cells. In this study, we show that an adenoviral vector (Ad.DF3-E1) in which the DF3/MUC1 promoter drives expression of E1A selectively replicates in MUC1-positive breast cancer cells. We also show that Ad.DF3-E1 infection of human breast tumor xenografts in nude mice is associated with inhibition of tumor growth. In contrast to a replication-incompetent adenoviral vector that infects along the injection track, Ad.DF3-E1 infection was detectable throughout the tumor xenografts. To generate an Ad.DF3-E1 vector with the capacity for incorporating therapeutic products, we inserted the cytomegalovirus (CMV) promoter upstream of the TNF cDNA. Infection with Ad.DF3-E1/CMV-TNF was associated with selective replication and production of TNF in cells that express MUC1. Moreover, treatment of MUC1-positive, but not MUC1-negative, xenografts with a single injection of Ad.DF3-E1/CMV-TNF was effective in inducing stable tumor regression. These findings demonstrate that the DF3/MUC1 promoter confers competence for selective replication of Ad.DF3-E1 in MUC1-positive breast tumor cells, and that the antitumor activity of this vector is potentiated by integration of the TNF cDNA.

Transcriptional and posttranscriptional regulation of macrophage-specific colony stimulating factor gene expression by tumor necrosis factor. Involvement of arachidonic acid metabolites.

The effects of tumor necrosis factor (TNF) on the regulation of macrophage-specific colony stimulating factor (CSF-1) gene expression have been studied in HL-60 cells during monocytic differentiation. CSF-1 transcripts were undetectable in uninduced HL-60 cells, reached maximal levels by 3 h of exposure to TNF, and returned to that of control cells by 24 h. Transcriptional run-on analysis demonstrated that exposure to TNF stimulated the rate of CSF-1 gene transcription by 6.4-fold. The combination of a protein synthesis inhibitor, cycloheximide, and TNF increased levels of CSF-1 mRNA compared with treatment by TNF alone. We also studied the signal transduction mechanisms responsible for regulating TNF-induced CSF-1 mRNA levels. Both 4-bromophenacyl bromide and quinacrine, inhibitors of phospholipase A2 activity, blocked TNF-induced increases in CSF-1 transcripts in a concentration-dependent manner, while caffeic acid and nordihydroguaiaretic acid, inhibitors of the 5-lipoxygenase pathway, had no detectable effect on induction of CSF-1 RNA. PGE2 or dibutyryl cAMP treatment of HL-60 cells in the presence of TNF blocked the expression of CSF-1 mRNA in a dose-dependent manner. These findings suggest that the increase in CSF-1 RNA observed during TNF treatment is regulated, at least in part, by both transcriptional and posttranscriptional mechanisms, and that PGE2 and cAMP regulate transcriptional activation of the CSF-1 gene by TNF.