NGR-TNF, a novel vascular-targeting agent, does not induce cytokine recruitment of proangiogenic bone marrow-derived cells.

BACKGROUND: Administration of certain chemotherapy drugs at the maximum tolerated dose, vascular-disrupting agents (VDAs) and irradiation can induce mobilisation and tumour homing of proangiogenic bone marrow-derived cells (BMDCs). Increases in cytokines and chemokines contribute to such mobilisation that eventually promotes tumour (re)growth. NGR-TNF is a vascular-targeting agent in advanced clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with tumour necrosis factor-alpha (TNF). We investigated whether NGR-TNF mobilises host BMDCs and growth factors. METHODS: Blood was obtained from Lewis lung carcinoma and 4T1 tumour-bearing mice at different time points following NGR-TNF, VDA or anti-VEGFR2/flk-1 antibody treatment. Levels of circulating growth factors were assessed by ELISAs. BMDCs were characterised by FACS. Circulating endothelial progenitor cells were defined as CD45(-)/CD13(+)/flk-1(+)/CD117(+)/7AAD(-), Tie2-expressing monocytes as CD45(+)/CD11b(+)/Tie2(+) and myeloid-derived suppressor cells as CD45(+)/CD11b(+)/Gr1(+) cells. RESULTS: NGR-TNF decreases tumour blood vessel density-inducing apoptosis of tumour and tumour endothelial cells. Unlike VDAs, or high-dose NGR-TNF, lower doses of NGR-TNF, comparable to those used in clinical trials, neither mobilise nor recruit to the tumour site proangiogenic BMDCs or induce host growth factors. CONCLUSION: Low-dose NGR-TNF exerts antitumour activity without inducing proangiogenic host responses, conceivably important for preventing/overcoming resistance and designing combination therapeutic strategies.

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.

Protective effect of the insulin-like growth factor I receptor on apoptosis induced by okadaic acid.

Okadaic acid (OKA), a potent inhibitor of serine phosphatases at concentrations as low as 20-25 nM, induces apoptosis of R- mouse embryo fibroblasts, which are 3T3-like cells devoid of type 1 insulin-like growth factor receptors (IGF-IRs). From R- cells, we have generated (by stable transfection) cell lines with IGF-IR numbers ranging from 0 (R- cells) to >10(6) receptors per cell. The wild-type IGF-IR protects R- cells from OKA-induced apoptosis, its protective effect being exquisitely dependent on the number of receptors. A small increment in wild-type receptor number (from 15 x 10(3) to 22 x 10(3) receptors/cell) is sufficient to change R(-)-derived cells from sensitive to resistant to apoptosis. We have also studied the effect of various mutations of the IGF-IR on its ability to protect R(-)-derived cells from OKA-induced apoptosis. Our data indicate a correlation between protection from apoptosis and the ability of the receptor to respond to insulin-like growth factor I with mitogenesis.

Grb10: A new substrate of the insulin-like growth factor I receptor.

Using the yeast two-hybrid system, we have isolated a new substrate of the insulin-like growth factor I receptor (IGF-IR), identified as Grb10, a member of the family of SH2 domain proteins. With the help of several mutants of the IGF-IR, we have mapped the binding site of Grb10 between amino acids 1229 and 1245 of the receptor, a sequence that is dispensable for the mitogenic activity of the IGF-IR. Grb10 coprecipitates with the IGF-IR in cell lysates and is probably involved in the regulation of its activity.

Biological activities and signaling pathways of the granulin/epithelin precursor.

Growth-regulated cells, such as 3T3 mouse embryo fibroblasts (MEFs), require more than one growth factor for growth, usually the insulin-like growth factor I (IGF-I) in combination with either platelet-derived growth factor or epidermal growth factor. Singly, these growth factors cannot sustain the growth of 3T3 cells. However, if the IGF-I receptor (IGF-IR) is even modestly overexpressed, then IGF-I, by itself, stimulates the growth of MEFs in monolayer and makes them capable of forming colonies in soft agar. The granulin/epithelin precursor (GEP) has been identified as the only growth factor, thus far, that can stimulate by itself the growth of R- cells, a 3T3-like cell line in which the genes for the IGF-IR have been deleted. We have expressed GEP in R- cells and show that these cells can now grow in serum-free medium. GEP, however, cannot replace other functions of the IGF-IR, such as protection from apoptosis (anoikis) or transforming activity (colony formation in soft agar). GEP activates, in R- cells, the two signaling pathways that are known to be sufficient for IGF-I-mediated mitogenesis in cells overexpressing the IGF-IR, the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways. This may explain why GEP, by itself, can replace the IGF-IR for growth in monolayer cultures. It also confirms that, for transformation, other pathways must be activated besides the two pathways that are sufficient for mitogenesis.

Insulin-like growth factor I receptor signaling in differentiation of neuronal H19-7 cells.

The type I insulin-like growth factor receptor (IGF-IR) is known to send two seemingly contradictory signals inducing either cell proliferation or cell differentiation, depending on cell type and/or conditions. H19-7 cells are rat hippocampal neuronal cells immortalized by a temperature-sensitive SV40 large T antigen that grow at 34 degrees C in epidermal growth factor or serum but differentiate at 39 degrees C when induced by basic fibroblast growth factor. At 39 degrees C, expression of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I-dependent differentiation. We have investigated the domains of the IGF-IR required for differentiation of H19-7 cells. The tyrosine 950 residue and serines 1280-1283 in the COOH terminus of the receptor are required for IGF-I-induced differentiation at 39 degrees C, although they are dispensable for IGF-I-mediated growth at 34 degrees C. Both domains have to be mutated to inactivate the differentiating function. The inability of these mutant receptors to induce differentiation correlates with mitogen-activated protein kinase activation. In contrast, inhibitors of phosphatidylinositol 3'-kinase have no effect on IGF-I-mediated differentiation of H19-7 cells, although they do inhibit the mitogenic response.

The role of the insulin-like growth factor I receptor in the transformation by simian virus 40 T antigen.

Balb/c 3T3 cells transformed by the tsA58 temperature-sensitive (ts) mutant of SV40 large T antigen, BalbA58 cells, grow in 1% serum at the permissive temperature of 34 degrees C but fail to grow at the restrictive temperature of 39.6 degrees C. Although incapable of growing, BalbA58 cells, in low serum at 39.6 degrees C, still synthesize DNA and tend to accumulate in the G2 phase of the cell cycle. Growth in 1% serum at 39.6 degrees C resumes if BalbA58 cells are treated with insulin-like growth factor I (IGF-I). By using cells overexpressing the IGF-I receptor, and cells with a targeted disruption of the IGF-I receptor genes, we show that: 1) the activation of the IGP-I receptor by its ligand(s) plays a major role in the ability of the SV40 large T antigen to promote growth in low serum; and 2) the IGF-I receptor plays a role in the progression of cells not only through G1, but also through the S and G2 phases of the cell cycle. These findings, together with other recent findings from the literature, suggest that one of the mechanisms by which oncogenes and tumor suppressor genes regulate cell growth is through the modulation of growth factors and their receptors.

Anti-apoptotic signaling of the IGF-I receptor in fibroblasts following loss of matrix adhesion.

The type 1 insulin-like growth factor receptor (IGF-IR) is known to protect cells from a variety of apoptotic injuries. In several instances, the anti-apoptotic effect of the wild type IGF-IR is more evident under conditions of anchorage-independence than in cells in monolayer cultures. We have investigated IGF-IR signaling in cells in anoikis, a form of apoptosis that occurs when cells are denied attachment to the extra-cellular matrix. IGF-I protects mouse embryo fibroblasts (MEF) from anoikis caused by withdrawal of growth factors. Survival is dependent on the concentration of IGF-I and a sufficient number of functional IGF-I receptors. In this model, IGF-I protection correlates best with ras activation and cell-to-cell aggregation, while PI3-kinase, Akt and MAP kinases seem to play a lesser, alternative role.

The role of the insulin receptor substrate-1 in the differentiation of rat hippocampal neuronal cells.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras.

32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of fibroblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We find that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic differentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in differentiation capacity. Over-expression of Shc proteins, by itself, promotes differentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in differentiation (v-Ha-Ras) is coupled with another differentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation.

The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.

The insulin-like growth factors (IGFs) are important mitogens that exert their proliferative effects on cells via the type I IGF receptors (IGF-R). The IGFs also associate with IGF binding proteins (IGFBPs). IGF-inhibitory, IGF-stimulatory, and IGF-independent effects of IGFBPs on cell growth have been reported. We have asked whether the IGFBP-3 has an effect on cell growth, which is independent of its ability to bind IGF-I and thus inhibit the activation of the IGF-I receptor. For this purpose, we have used a fibroblast cell line (R- cells) derived from mouse embryos homozygous for a targeted disruption of the IGF-R gene. When compared with wild type cells (W), which bind IGF-I with high affinity and specificity, R- cells transfected with a mammalian expression vector containing the human (h) IG-FBP-3 cDNA were selected (R-/BP3) and found to express hIGFBP-3 mRNA (detected by Northern blots) and to secrete hIGFBP-3 protein [detected by Western ligand blotting (WLB), immunoblotting, and immunoprecipitation as well as immunofluorescence confocal microscopy]. Growth of five different R- cells, and 10-fold slower compared with W cells, grown under identical conditions. Confluent R- cells. These experiments show that the overexpression of IGFBP-3 has an inhibitory effect on cell growth which does not involve IGF binding to, or signal transduction via, the type I IGF-R. We conclude that the cellular production of IGFBPs serves as a negative regulator of cell proliferation which involves a cellular signaling pathway separate from the IGF-IGF-R system.

Regulation of Id2 gene expression by the type 1 IGF receptor and the insulin receptor substrate-1.

The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70(S6K), a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70(S6K). Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.

Biological characterisation of primary and metachronous lesions in breast cancer patients.

Proliferative activity, evaluated as [3H]thymidine labelling index ([3H]dT LI), and hormone receptors were determined on 97 primary breast cancers and on metachronous lesions from the same patient. Overall, the [3H]dT LI of metachronous lesions was significantly higher than that of the primary tumour (P = 0.003). Hormone receptor profiles of the two lesions were similar in about 75% of the cases; disagreements were mainly due to a disappearance of hormone receptors in metachronous lesions. In contralateral tumours, [3H]dT LI and hormone receptors were unrelated to those of the relative primary lesion. In this series of relapsing patients, [3H]dT LI was unrelated to hormone receptor status in the primary tumour, but it was higher in the metachronous lesions from patients with hormone receptor-negative primary tumours. For patients given no systemic therapy between surgery and relapse, the time to develop local-regional recurrences or contralateral tumours was inversely related to the [3H]dT LI of the metachronous lesions.

p53 expression, DNA content and cell proliferation in primary and synchronous metastatic lesions from ovarian surface epithelial-stromal tumours.

The aim of this study was to investigate biological heterogeneity between primary and metastatic ovarian cancer lesions from individual patients as a means of elucidating steps in clinical progression. Cancer tissue from 61 untreated patients with ovarian surface epithelial-stromal tumours was examined. p53 expression detected immunocytochemically by the PAb1801 antibody, DNA content evaluated by flow cytometry, and cell proliferation evaluated as the [3H]thymidine labelling index were investigated in primary tumours and corresponding synchronous metastases. The frequency of p53 positivity was similar in primary (62%) and metastatic (66%) sites, with an agreement between the two lesions from the same patient in 97% of the cases. Similarly, aneuploidy frequency (80%) and DNA indices were superimposable in primary and metastatic lesions from the same patient, with a 94% agreement. The frequency of aneuploidy was higher in p53-positive than in p53-negative lesions. An overall poor agreement (rs = 0.44) was observed for proliferative activity of primary and metastatic lesions, due to a heterogeneous profile in omental with respect to primary tumours, which was mainly evident in p53-positive cancers. Conversely, cell proliferation of peritoneal, abdominal and pelvic lesions was qualitatively similar to that of the primary tumour in 88% of patients.

Potentials of cell kinetics in the management of patients with ovarian cancers.

The relevance of 3H-thymidine labeling index (3H-dt LI) on clinical outcome was evaluated on 85 patients with advanced ovarian cancers treated with carboplatin or cisplatin alone (39 cases) or cisplatin in association with doxorubicin and/or cyclophosphamide (46 cases). 3H-dT LI of the primary tumour was significantly related to the 3-year probability of survival in patients treated by monochemotherapy (low LI, 63%; high LI, 21%; P = 0.013) but not in those treated with polychemotherapy. Analysis of the relation between cell kinetics and clinical outcome as a function of treatment showed that in patients with rapidly proliferating tumours the 3-year survival was significantly higher following polychemotherapy than monochemotherapy (51 vs. 21%; P = 0.04). In patients with slowly proliferating tumours no significant difference in survival was observed following the two types of treatment for the overall series, whereas in patients not achieving a complete response survival was significantly higher following monochemotherapy than polychemotherapy (61 vs. 9%; P = 0.008).

Detection of DNA polymerase-beta gene expression by competitive polymerase chain reaction in human ovarian carcinoma cells.

The reliability of the competitive polymerase chain reaction (competitive PCR) for the detection and quantitation of gene expression in small tumor samples was evaluated. DNA polymerase-beta gene expression was detected in human ovarian cancer cell lines displaying a different degree of cisplatin resistance. The level of DNA polymerase-beta cDNA in the resistant cell line was threefold that of the parental sensitive line. Our results indicate that competitive PCR is a reproducible and sensitive method to detect differences in gene expression in small samples and open the possibility of using this approach to detect DNA polymerase beta cDNA in small samples from clinical tumors.

The IGF-I receptor protects tumor cells from apoptosis induced by high concentrations of serum.

High concentrations of fetal bovine serum (FBS) induce apoptosis of cells in culture. A decrease in the number of insulin-like growth factor I receptors (IGF-IR) sensitizes the cells to serum-induced apoptosis. Autophosphorylation of the IGR-IR is not affected by high serum. These results may explain why the targeting of the IGF-IR renders tumor cells much more sensitive to apoptosis in vivo than in vitro.

IGF-I receptor signalling in transformation and differentiation.

The type 1 insulin-like growth factor receptor (IGF-IR) sends several signals, some of which are contradictory. When the concentrations of insulin receptor substrate 1 (IRS-1), a major substrate of the IGF-IR, are high, the signal is mitogenic, anti-apoptotic, and can even cause malignant transformation. However, in the absence of IRS-1, the IGF-IR sends a differentiation signal, which leads to granulocytic differentiation in haemopoietic cells. The mitogenic signal of the IGF-IR/IRS-1 combination depends largely, but not exclusively, on the activation of the phosphatidylinositol-3 kinase (PI3K).

Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGF-independent mechanism.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is known to block IGF action and inhibit cell growth. IGFBP-3 is thought to act by sequestering free IGFs or, possibly, act via a novel IGF-independent mechanism. Supporting its role as a primary growth inhibitor, IGFBP-3 production has been shown to be increased by cell growth-inhibitory agents, such as transforming growth factor-beta (TGF-beta), and the tumor suppressor gene p53. In this paper, we demonstrate, for the first time, a novel function of IGFBP-3 as an apoptosis-inducing agent and show that this action is mediated through an IGF.IGF receptor-independent pathway. In the p53 negative prostate cancer cell line, PC-3, the addition of recombinant IGFBP-3 resulted in a dose-dependent induction of apoptosis. 125I-IGFBP-3 bound with high affinity to specific proteins in PC-3 cell lysates and plasma membrane preparations. These membrane-associated molecules may serve as receptors that mediate the direct effect of IGFBP-3 on apoptosis. In addition, in an IGF receptor-negative mouse fibroblast cell line, treatment with recombinant IGFBP-3 as well as transfection of the IGFBP-3 gene induced apoptosis, suggesting that neither IGFs nor IGF receptors are required for this action. Furthermore, treatment with TGF-beta1, a known apoptosis-inducing agent, resulted in the induction of IGFBP-3 expression 6-12 h before the onset of apoptosis. This effect of TGF-beta1 was prevented by co-treatment with IGFBP-3-neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. These findings suggest that IGFBP-3 induces apoptosis through a novel pathway independent of either p53 or the IGF.IGF receptor-mediated cell survival pathway and that IGFBP-3 mediates TGF-beta1 induced apoptosis in PC-3 cells.