Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor in malignant astrocytomas.

PURPOSE: Preclinical animal experiments support the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor (IGF-IR/AS ODN) as an effective potential antitumor agent. We performed a human pilot safety and feasibility study using an IGF-IR/AS ODN strategy in patients with malignant astrocytoma. PATIENTS AND METHODS: Autologous glioma cells collected at surgery were treated ex vivo with an IGF-IR/AS ODN, encapsulated in diffusion chambers, reimplanted in the rectus sheath within 24 hours of craniotomy, and retrieved after a 24-hour in situ incubation. Serial posttreatment assessments included clinical examination, laboratory studies, and magnetic resonance imaging scans. RESULTS: Other than deep venous thrombosis noted in some patients, no other treatment-related side effects were observed. IGF-IR/AS ODN-treated cells, when retrieved and assessed, were < or = 2% intact by trypan blue exclusion, and none of the intact cells were viable in culture thereafter. Parallel Western blots disclosed IGF-IR downregulation to < or = 10% after ex vivo antisense treatment. At follow-up, clinical and radiographic improvements were observed in eight of 12 patients, including three cases of distal recurrence with unexpected spontaneous or postsurgical regression at either the primary or the distant intracranial site. CONCLUSION: Ex vivo IGF-IR/AS ODN treatment of autologous glioma cells induces apoptosis and a host response in vivo without unusual side effects. Subsequent transient and sustained radiographic and clinical improvements warrant further clinical investigations.

Antitumor effects elicited by antisense-mediated downregulation of the insulin-like growth factor I receptor (review).

Antisense-mediated downregulation of the insulin-like growth factor I receptor results in massive apoptosis of tumor cells in vivo, leading to abrogation of tumorigenesis. In addition to the apoptotic effect, antitumor responses are elicited in syngeneic immunocompetent animals, protecting them from subsequent tumor challenge and causing regression of established tumors with no further recurrence. The mechanisms involved in the antitumor responses elicited in the animals following exposure to antisense cells are discussed, focusing on the immunogenicity of the antisense cells as well as the effectors that participate in these responses.

Inhibition of tumorigenesis and induction of apoptosis in human tumor cells by the stable expression of a myristylated COOH terminus of the insulin-like growth factor I receptor.

The insulin-like growth factor I receptor (IGF-IR) plays an important role in cell transformation and in protection from apoptosis. Although the wild-type IGF-IR generally has an antiapoptotic effect, there are reports that its COOH terminus may actually generate a proapoptotic signal. Three different expression plasmids, all coding for the COOH-terminal sequences of the human IGF-IR, MyCF, CF, and MyKCF, were stably transfected into human ovarian carcinoma CaOV-3 cells. All three plasmids had no effect on monolayer growth but strongly inhibited colony formation in soft agar. Only one of the plasmids, MyCF, expressing the last 112 amino acids of the IGF-IR and carrying a myristylation signal, caused large-scale apoptosis of CaOV-3 cells in vivo and abrogation of tumorigenesis in nude mice. The plasmid expressing the MyCF sequence was also introduced into human glioblastoma T98G cells, where it decreased the clonogenicity of cells, caused a marked inhibition of colony formation in soft agar, and induced apoptosis in vivo. A double mutation at residues 1293 and 1294 of MyCF completely abrogated its inhibitory and proapoptotic activities. Neither the autophosphorylation of the IGF-IR nor the tyrosyl phosphorylation of IRS-1 was affected by the expression of the MyCF plasmid. These and other findings suggest that a stably expressed myristylated COOH terminus of the IGF-IR can induce apoptosis in human tumor cells in vivo and inhibit tumorigenesis in nude mice by a mechanism that avoids the protective effect of the IGF-IR.

The baculovirus anti-apoptotic p35 protein promotes transformation of mouse embryo fibroblasts.

The baculovirus p35 protein is a potent inhibitor of programmed cell death induced by a variety of stimuli in insects, nematodes, and mammalian cell lines. The broad ability of p35 in preventing apoptosis has led us to investigate its effect on mouse embryo fibroblasts in vitro and in vivo. For this purpose, we have used R- cells (3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes) and R508 cells (derived from R- and with 15 x 10(3) IGF-IRs per cell). Both cell lines grow normally in monolayer, but they do not form colonies in soft agar, and they are non-tumorigenic in nude mice. We show here that, in addition to its anti-apoptotic effect, p35 causes transformation of R508 cells, as evidenced by the following: 1) decreased growth factor requirements, 2) ability to form foci in monolayer and colonies in soft agar, and 3) ability to form tumors in nude mice. Since R- cells stably transfected with p35 do not transform, our observations suggest that in addition to its effect as an inhibitor of apoptosis, the baculovirus p35 protein has transforming potential that requires the presence of the IGF-IR. The possibility that these two properties could be separated was confirmed by demonstrating that R508 cells expressing another anti-apoptotic protein, Bcl-2, could not form tumors in nude mice.

A novel class of peptides that induce apoptosis and abrogate tumorigenesis in vivo.

In testing the ability of certain synthetic peptides of biological origin to bind to cell surfaces, we unexpectedly found that they could induce apoptosis and inhibit tumorigenesis in rodent and human tumor cell lines. In vitro pre-incubation with the peptides at concentrations as low as 10(-12) M inhibited tumorigenesis in nude mice, in a dose-dependent fashion. The inhibition of tumorigenesis was reflected in the rapid induction of apoptosis of tumor cells, pre-incubated with the peptides, and tested under conditions of anchorage-independence. Induction of apoptosis was detectable even at concentrations of 10(-12)-10(-13) M (0.1-1.0 pM). Aspecific toxicity of the synthetic peptides was ruled out by the demonstration that single amino acid substitutions (in at least 4 peptides) completely abrogated the pro-apoptotic effect, even at a concentration of 10(-5) M (10 microM).

The role of mGrb10alpha in insulin-like growth factor I-mediated growth.

Several isoforms of Grb10 are known to interact with either the insulin receptor or the insulin-like growth factor I (IGF-I) receptor, or both. Inasmuch as the data in the literature on the function of Grb10 are not always concordant, we have investigated the role of one of these isoforms, mGrb10alpha, in cell proliferation. For this purpose, a plasmid expressing mGrb10alpha was stably transfected into p6 cells and other mouse embryo fibroblast cell lines. An overexpressed mGrb10alpha inhibits IGF-I-mediated growth, causes a delay in the S and G2 phases of the cell cycle, and partially reverses the transformed phenotype. In contrast, it has no effect on insulin stimulation of cell proliferation. These studies indicate that this isoform of Grb10 has an inhibitory effect on IGF-I signaling of cell proliferation.

Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA.

We have previously shown that C6 cells expressing an antisense insulin-like growth factor I receptor (IGF-IR) RNA are no longer tumorigenic in syngeneic rats, protecting them from subsequent subcutaneous tumor challenge and causing regression of established subcutaneous tumors. In the present study, we have investigated the efficacy of this strategy on intracerebrally implanted C6 rat glioblastoma cells. We demonstrate that C6 cells expressing an antisense IGF-IR RNA implanted for 24 h in the subcutaneous tissue of the rats are able to elicit an anti-tumor response in the brain, leading to complete brain tumor regression and long-term survival of the rats. These findings suggest the possibility of therapeutic intervention in human gliomas.

A soluble insulin-like growth factor I receptor that induces apoptosis of tumor cells in vivo and inhibits tumorigenesis.

By a frame-shift mutation, we have engineered a human IGF-I receptor (IGF-IR) cDNA that produces a receptor 486 amino acids long (plus the 30 amino acids of the signal peptide). This receptor, which we have designated as 486/STOP, is partially secreted into the medium of cells in culture and markedly inhibits the autophosphorylation of the endogenous IGF-IRs as well as the activation of the signaling pathway. The 486/STOP receptor acts as a strong dominant negative for several growth functions: (a) it inhibits the growth of cells in monolayers; (b) it inhibits the growth of transformed cells in soft agar; (c) it induces extensive apoptosis in vivo; and (d) it inhibits tumorigenesis in syngeneic rats. This is the first demonstration that a dominant negative of the IGF-IR can induce massive apoptosis of tumor cells in vivo.

Inhibition of hamster mesothelioma tumorigenesis by an antisense expression plasmid to the insulin-like growth factor-1 receptor.

We evaluated the effect of antisense insulin-like growth factor (IGF) receptor transcripts on the proliferation and tumorigenicity in an SV40-induced, immunocompetent hamster mesothelioma model (H9A). Expression of IGF-1 and IGF-1 receptor (IGF-1R) genes was identified from H9A RNA using reverse transcription-PCR and Northern analysis. H9A cells were electroporated with inducible expression vectors (under the transcriptional control of heat shock promoter HSP70) containing a cDNA fragment corresponding to base pairs 1-309 of IGF-1R in the sense or antisense orientation to generate the respective clones A3 sense or B9 antisense. The expression vector in genomic DNA was detected with PCR analysis as a 173-bp fragment on ethidium bromide gels. The effects of the expression vectors were then evaluated in vitro under active (at 39 degrees C) or inactive (at 34 degrees C) conditions. At 39 degrees C, the B9 antisense transfectants demonstrated significantly less proliferation than A3 sense transfectants (P2 < 0.02). At 34 degrees C, cell growth of A3 sense- and B9 antisense-transfected cells was not significantly different. In vivo tumorigenicity was evaluated in hamsters inoculated with 10(5) A3 sense- or B9 antisense-transfected cells. The A3 sense clones resulted in greater numbers of tumors in vivo compared to the B9 antisense clone (P2 = 0.0001). When genomic DNA from tumors that developed in A3 sense and B9 antisense animals was analyzed for the expression vectors, a 173-bp fragment amplified from the expression vector was identified in the sense tumors but not in antisense B9 or wild-type H9A tumors, indicating a loss of the vector from the antisense clones that proliferated in vivo. The inhibitory effect of IGF-1R antisense transcripts on hamster mesothelioma demonstrated in this study by decreased growth and tumorigenicity in vitro and in vivo may have implications for the therapy of human mesothelioma.

Ethanol-induced inhibition of cell proliferation is modulated by insulin-like growth factor-I receptor levels.

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-I receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem. 268:21777-21782 (1993)]. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-I receptor (p6 cells) was only partially inhibited by ethanol BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-I overcame the inhibition of IGF-I receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat giloblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated in cells expressing antisense mRNA to the IGF-I receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/IGF-I receptor autocrine pathway. Indeed, EGF stimulated an increase in IGF-I receptor levels by more than 100%. Ethanol completely inhibited the proliferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I-receptor, ethanol has no direct effect on EGF receptor activation.

Effect of mutations at serines 1280-1283 on the mitogenic and transforming activities of the insulin-like growth factor I receptor.

The insulin-like growth factor I receptor (IGF-IR) controls the extent of cell proliferation in a variety of cell types by at least 3 different ways: it is mitogenic, it causes transformation, and it protects cells from apoptosis. Previous reports indicated that certain domains in the C terminus of the IGF-IR transmitted a transforming signal that is additional to and separate from the mitogenic signal. We have now mutated the four serine residues at 1280-1283 of the IGF-IR, and transfected the mutant receptor into R- cells. Cells expressing the mutant receptor are fully responsive to IGF-I mediated mitogenesis, but are not transformed (no colony formation in soft agar). Several downstream signal transducers are not affected by the mutation, again suggesting a separate pathway for transformation. The mutant receptor can act as a dominant negative for growth, but cannot induce apoptosis in cells with endogenous wild-type receptors.

Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor I receptor antisense mRNA.

The expression of insulin-like growth factor I receptor (IGF-IR) antisense mRNA inhibits the growth of C6 rat glioblastoma cells both in vitro and in vivo [Cancer Res (1994) 54:2218]. Moreover, the injection of C6 cells expressing an antisense mRNA to the IGF-IR into syngeneic rats prevents subsequent wild-type tumorigenesis and induces regression of established tumors. For the study of immune function in syngeneic rats, C6 cells expressing either IGF-IR sense or IGF-IR antisense mRNA were injected and splenic lymphocyte function analyzed in vitro after 2 weeks. Cytotoxic, CD8+ lymphocytes from animals injected with IGF-IR antisense cells, but not from those treated with IGF-IR sense cells, proliferated in vitro in response to wild-type C6 cells. Wild-type C6 cells or IGF-IR-sense-RNA-expressing cells rapidly formed tumors upon subcutaneous injection into athymic nude mice. IGF-IR antisense cells were weakly tumorigenic, exhibiting a six- to tenfold increase in tumor latency. Injection of IGF-IR antisense C6 cells mildly delayed the development of wild-type tumors, and did not induce the regression of established wild-type C6 tumors in athymic nude mice. Thus, these findings demonstrate the stimulation of a cellular immune response in rats following the injection of IGF-IR antisense cells. However, studies of athymic nude mice indicate that expression of IGF-IR antisense mRNA also inhibits C6 cells tumorigenicity by additional mechanisms.

Mutant IGF-I receptors as dominant negatives for growth and transformation.

The insulin-like growth factor I receptor (IGF-IR) plays a crucial role in cell growth, transformation and protection from apoptosis. We have transfected several mutant IGF-IRs into C6 rat glioblastoma cells, in order to determine whether they can act as dominant negatives. We find that some of them can act as dominant negatives in growth assays (monolayer or soft agar), but that none of those examined can induce apoptosis in C6 cells.

Correlation between apoptosis, tumorigenesis, and levels of insulin-like growth factor I receptors.

We have investigated whether there is a quantitative relationship between the insulin-like growth factor I receptor (IGF-IR), the extent of apoptosis in vivo, and tumorigenesis. C6 rat glioblastoma cells were treated with increasing concentrations of antisense oligodeoxynucleotides to the IGF-IR RNA. The extent of apoptosis in vivo is correlated to the decrease in IGF-IR levels and, in turn, tumorigenesis in nude mice is correlated to the fraction of surviving cells. In syngeneic rats, a host response leads to complete inhibition of tumorigenesis. These findings establish, for the first time on a quantitative basis, the relationship between IGF-IR levels and the extent of apoptosis, as well as the relationship between the initial apoptotic event and the time of appearance of transplantable tumors.

The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo.

The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.

Ethanol inhibits insulin-like growth factor-1-mediated signalling and proliferation of C6 rat glioblastoma cells.

BACKGROUND: Alcohol consumption during pregnancy often results in disorders of fetal development (Fetal Alcohol Syndrome). The brain appears to be particularly vulnerable, and alcohol abuse during pregnancy is probably the most common cause of acquired mental retardation. We therefore studied the in vitro effects of ethanol on insulin-like growth factor-1 (IGF-1)-mediated proliferation of rat C6 glioblastoma cells. EXPERIMENTAL DESIGN: The proliferation of C6 rat glioblastoma cells was measured in serum-free medium supplemented with specific growth factors in the presence or absence of ethanol. The effect of ethanol on IGF-1 receptor and insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation was determined by immunoprecipitation and Western blotting, as was the phosphatidylinositol 3-kinase content within IRS-1 immunoprecipitates. RESULTS: C6 cells grew slowly in serum-free medium and proliferated in response to IGF-1. Ethanol, at physiologically tolerated concentrations, markedly inhibited the growth of C6 cells in response to IGF-1, but had no effect on the proliferative rate in the presence of platelet-derived growth factor or 1% fetal bovine serum. Inhibition of cell proliferation was evident when ethanol was only present during a 1-hour pulse of IGF-1. Cell growth in the presence of IGF-2 was also prevented by ethanol. The inhibition of IGF-1-mediated cell proliferation was accompanied by abrogation of IGF-1 receptor tyrosine autophosphorylation. Ethanol also interfered with the IGF-1-induced tyrosine phosphorylation of IRS-1, and the association of phosphatidylinositol-3 kinase with IRS-1. CONCLUSIONS: The data indicate that physiologically relevant concentrations of ethanol inhibit the responses of glial cells to IGF-1, including IGF-1 receptor autophosphorylation, IRS-1 and phosphatidylinositol-3 kinase activation, and cell growth.

Growth inhibition of human melanoma cells in nude mice by antisense strategies to the type 1 insulin-like growth factor receptor.

The growth of human melanoma cells FO-1 in nude mice is strongly inhibited or even abrogated when the cells are stably transfected with a plasmid expressing an antisense RNA to the insulin-like growth factor 1 receptor (IGF-1R) RNA, which causes a marked reduction in the number of IGF-1 receptors. When a tumor arises after a long delay in nude mice, it can be shown that the tumor cells have lost the expression plasmid and that the number of IGF-1 receptors has returned to wild-type levels. The antisense effect is even more remarkable, since the growth of FO-1 melanoma cells in monolayers is not affected by the expression of the antisense RNA. Inhibition of tumorigenesis was also evident when FO-1 melanoma cells were treated with antisense oligodeoxynucleotides to the IGF-1R RNA prior to injection into nude mice. These results confirm in human cells that the IGF-1R plays a dominant role in transformation and tumorigenesis and that its effect on tumorigenesis is more profound than its effect on mitogenesis.