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.

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.

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.

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.

Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce regression of wild-type tumors.

Insulin-like growth factor-1 (IGF-1) and IGF-2 are critical regulators of cell proliferation. The growth-promoting action of both ligands is mediated by the type 1 IGF receptor (IGF-1R). We have investigated the role of the IGF-1R in the growth and tumorigenicity of rat C6 glioblastoma cells. For this purpose, antisense RNA to IGF-1R RNA was introduced into cells by either the addition of oligodeoxynucleotides or by transfection with plasmids that express antisense RNA to IGF-1R RNA. At low cell density, C6 cells grew slowly in serum-free medium and proliferated with the sole addition of IGF-1 or IGF-2. Both antisense IGF-1R oligodeoxynucleotides and stable transfection with a plasmid expressing an antisense IGF-1R RNA inhibited IGF-1-mediated growth in monolayers and clonogenicity in soft agar. Sense oligodeoxynucleotides and sense-expressing plasmid had no effect on either parameter. In stable antisense transfectants, tyrosine-phosphorylated IGF-1 receptors were not detectable, although they were easily detected in wild-type cells. When wild-type C6 cells were injected s.c. into syngeneic immunocompetent rats, tumors developed within 1 week. In contrast, stably transfected C6 cells overexpressing antisense IGF-1R RNA were nontumorigenic. Moreover, when C6 IGF-1R antisense cells were injected, subsequent tumor formation by wild-type C6 cells was completely prevented. Finally, injection of C6 IGF-1R antisense cells into rats carrying an established wild-type C6 tumor caused complete regression of the tumors. The results demonstrate the critical importance of the IGF-1R in glioblastoma cell growth, clonogenicity, and tumorigenicity. Although the mechanism is presently unknown, the fact that the injection of C6 cells expressing an antisense RNA to IGF-1R RNA leads to regression of already established wild-type C6 tumors suggests the possibility of practical applications.

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.

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.

Subtyping of human immunodeficiency virus isolates with a panel of monoclonal antibodies: identification of conserved and divergent epitopes on p17 and p25 core proteins.

We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides' approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 11-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of p25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.

Clonal analysis of murine B cell response to the human immunodeficiency virus type 1 (HIV1)-gag p17 and p25 antigens.

The antigenicity of HIV-gag p17 and p25 proteins was analyzed using a panel of 52 monoclonal antibodies (mAb) derived from 17 independent fusion experiment protocols performed in 12 different laboratories. These mAb were tested for their capacity to bind peptides corresponding to sequences of HIV1-BRU-gag p17 and p25. Thirty-five overlapping peptides (P1 to P35) totally covering the p17 and p25 proteins were used. This study allowed us to identify four immunodominant regions inducing B cell response, two on p17 corresponding to P2 and P13 (amino acids 11-25 and 121-132, respectively) and two on p25 corresponding to P21 and P28-P29-P30 (a.a. 201-218 and 285-320 respectively). According to secondary structure predictions, peptides P2 and P21 contained hydrophilic alpha helix folded regions whereas P13 sequence presented a beta turn propensity. These regions and the P28-30 region were also predicted to be easily accessible to mAb. Several other p25-derived peptides: P15 (a.a. 142-156), P16 (a.a. 148-162), P19 (a.a. 176-192), P22 (a.a. 219-233) and P23 (a.a. 233-253) were recognized by mAb. No p17-derived peptide other than P2, P13 and P12 (a.a. 111-123) was found to react with mAb. Cross-blocking studies between mAb, suggested the existence of more than four distinct epitopic areas on p17 and eight on p25.

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.

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.

13-cis-retinal stimulates proliferation and induces intranuclear protein accumulation in the human mammary tumor cells MCF-7.

The human mammary tumor cells MCF-7 show enhanced proliferation when treated with low doses (10(-8)-10(-7) M) of 13-cis Retinal (a vitamin A derivative). These results are independent of the growth medium used. We describe a novel effect of 13-cis Retinal: the increased synthesis and accumulation of nuclear proteins in chronically treated cells. The cytoplasmic proteins and proteins released to the culture medium are transiently and oppositly modified. Moreover, chronic treated cells have growth advantages over the untreated counterparts in a clonogenic soft agar assay.

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.

Growth factors and hormones which affect survival, growth, and differentiation of the MCF-7 stem cells and their descendants.

The human breast tumor cell line was separated by Percoll density gradient centrifugation into six different subpopulations, A to F, one of which (E) appears to contain the stem cells on the basis of several criteria (M. Resnicoff et al. 1987, Proc. Natl. Acad. Sci. USA 84, 7295. We now analyzed the response of the isolated subpopulations to insulin, thrombin, PGF2 alpha, estradiol, and 13-cis-retinal. We demonstrate that the first two growth factors stimulate [3H]thymidine incorporation in the more differentiated subpopulations (D and F), while PGF2 alpha has mitogenic activity in subpopulations C and D. In the absence of any added growth factor, estradiol has the extreme and transient capacity of allowing the stem cell to detach from the tissue culture dish and to grow in suspension as multicellular aggregates (MCF-7/SE cells). 13-cis-Retinal acts as a negative modulator of differentiation and protects the cells from the inhibitory and differentiation activity of Na-butyrate.

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.

Insulin-like growth factor-1 and its receptor mediate the autocrine proliferation of human ovarian carcinoma cell lines.

BACKGROUND: IGF-1 regulates the growth of diverse mammalian cell types including several human carcinoma cell lines. The IGF-1 receptor is a glycosylated heterodimer which, upon binding with IGF-1, undergoes tyrosine autophosphorylation. The autophosphorylation of the beta-receptor subunit is a strict requirement for its mitogenic properties. EXPERIMENTAL DESIGN: In this study, we have assessed the role of the IGF-1 receptor in the proliferation of ovarian carcinoma cell lines in culture. Effects of anti-sense and sense oligodeoxynucleotides to IGF-1 receptor RNA were tested. RESULTS: The human ovarian carcinoma cell lines OVCAR-3 and CaOV-3 both grew autonomously in serum-free medium, and their growth was further stimulated by the addition of IGF-1. Incubation of cells with anti-sense oligodeoxynucleotides corresponding to the IGF-1 receptor RNA markedly inhibited cell proliferation both in serum-free medium and in the presence of IGF-1. The inhibition of cell growth by the oligodeoxynucleotides corresponded to a reduction in the amount of detectable phosphorylated IGF-1 receptor. CONCLUSIONS: The findings indicate that IGF-1 and its specific receptor mediate the autocrine proliferation of human ovarian carcinoma cell lines.

Characterization of monoclonal antibodies identifying type and strain-specific epitopes of human immunodeficiency virus type 1.

Several hybridoma cell lines were raised against the highly cytopathic Zaïrian isolate of Human Immunodeficiency Virus (HIV), HIV1-NDK. The specificity of the secreted monoclonal antibodies (mAb) was demonstrated by immunoblotting, radioimmunoprecipitation and immunofluorescence. Two hybridoma cell lines secreted mAb reacting with independent epitopes of the NDK p17 capsid protein and its precursors. One, RL16.24.5, is specific for the NDK isolate whereas the other, RL16.45.1, along with anti-p25 RL16.30.1 mAb, bind all HIV1 isolates but not HIV2. Together with the previously described mAb RL4.72.1 those reagents define lentivirus subfamily (HIV1, HIV2, SIV) type/subtype (HIV1) and strain (HIV1-NDK) specific epitopes expressed on HIV1-NDK core proteins. The last mAb RL16.76.1 binds the env gene products gp160 and gp120.

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.