Mutations in the Wilms' tumor gene WT1 in leukemias

The tissue-specific Wilms' tumor gene WT1 is expressed in a range of acute leukemias and hematopoietic cell lines. Using single-strand conformational polymorphism analysis, we have found mutations in the WT1 gene in 4 of 36 acute leukemias. WT1 mutations are found in 15% of cases of acute myeloid leukemia, in which they are associated with a poor response to chemotherapy. The mutations comprise small insertions in exons 1 and 7 and a nonsense mutation in exon 9. All are predicted to produce a truncated WT1 protein with absence or disruption of the zinc finger region. These are the first mutations in the WT1 gene to be described in sporadic leukemia.     

Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation

IGF-I is a key factor in intrauterine development and postnatal growth and metabolism. The secretion of IGF-I in utero is not dependent on GH, whereas in childhood and adult life, IGF-I secretion seems to be mainly controlled by GH, as revealed from studies on patients with GHRH receptor and GH receptor mutations. In a 55-yr-old male, the first child of consanguineous parents, presenting with severe intrauterine and postnatal growth retardation, microcephaly, and sensorineural deafness, we found a homozygous G to A nucleotide substitution in the IGF-I gene changing valine 44 into methione. The inactivating nature of the mutation was proven by functional analysis demonstrating a 90-fold reduced affinity of recombinantly produced for the IGF-I receptor. Additional investigations revealed osteoporosis, a partial gonadal dysfunction, and a relatively well-preserved cardiac function. Nine of the 24 relatives studied carried the mutation. They had a significantly lower birth weight, final height, and head circumference than noncarriers. In conclusion, the phenotype of our patient consists of severe intrauterine growth retardation, deafness, and mental retardation, reflecting the GH-independent secretion of IGF-I in utero. The postnatal growth pattern, similar to growth of untreated GH-deficient or GH-insensitive children, is in agreement with the hypothesis that IGF-I secretion in childhood is mainly GH dependent. Remarkably, IGF-I deficiency is relatively well tolerated during the subsequent four decades of adulthood. IGF-I haploinsufficiency results in subtle inhibition of intrauterine and postnatal growth.     

The International Reference Reagent for insulin-like growth factor-I

Three preparations of recombinant DNA-derived insulin-like growth factor-I (IGF-I) were obtained, prepared in ampoules coded 86/522, 86/720 or 87/518, and evaluated as candidate International Reference Reagents in an international collaborative study (nine laboratories in four countries) in response to a request by the World Health Organization (WHO). Immunoassay dose-response curves for each of the three preparations did not in general differ significantly from those of local standards or from those of ampouled preparations of serum-derived IGF-I which were included in the study. The estimates of ampoule contents in terms of local standards showed considerable heterogeneity; the between-laboratory variability of estimates in terms of local standards was ten times greater than the inherent variability of estimates from these systems as estimated from comparisons of coded duplicates. Bioassay data were limited, and those available were inconsistent with immunoassay data. Of the three preparations, ampoules coded 86/720 were derived from an IGF-I preparation that was heterogeneous by high-performance liquid chromatography, and stability data for the preparation 86/522 were anomalous. As a result, the ampouled preparation coded 87/518 has been established by WHO as the International Reference Reagent for IGF-I for immunoassay, with an assigned ampoule content of 3.1 micrograms/ampoule, and is available from the National Institute for Biological Standards and Control.     

Down-regulation of Leydig cell insulin-like growth factor-I gene expression by interleukin-1.

We have reported previously that insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) is expressed in Leydig cells and that IGF-I can enhance androgen production, whereas interleukin-1 (IL-1) is a potent inhibitor of Leydig cell steroidogenesis. Molecular cloning studies have confirmed the existence of at least two species of IL-1: IL-1 alpha and IL-1 beta. Both IL-1 alpha and beta bind to the same receptors and have the same spectrum of biological activities. The purpose of the present study was to elucidate the molecular mechanisms of the interaction between IGF-I and IL-1 both in vivo and in vitro. Adult Sprague-Dawley rats (55-65 days old) were treated with three injections of human recombinant IL-1 beta (1 microgram/rat ip) at 12-h intervals. Rats were killed 2 h after the last injection of IL-1 beta. Purified Leydig cells were isolated and RNA extracted for Northern blot analyses. For in vitro studies, highly purified Leydig cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 0.1% fetal calf serum with or without IL-1 beta (1-100 ng/ml) for 24 h. RNA was then extracted from these cells. For time course studies, purified Leydig cells were initially cultured for 24 h. Fresh medium was then added with or without IL-1 beta (10 ng/ml), and the cultures were continued for an additional 2, 4, or 6 h. In vivo administration of IL-1 beta inhibited IGF-I mRNA expression in Leydig cells (a 40% reduction, P less than 0.05). IL-1 beta also suppressed IGF-I mRNA expression in Leydig cells in vitro in a time- and dose-dependent fashion. Inhibitory effects of IL-1 beta (10 ng/ml) could be demonstrated as early as 2 h and reached a nadir at 6 h (a 60% reduction, P less than 0.05). IL-1 beta (100 ng/ml) inhibited IGF-I mRNA expression to about 10% of the controls (P less than 0.01). Moreover, the inhibitory effect of IL-1 beta could be reversed by the addition of IL-1 receptor antagonist. In conclusion, IL-1 beta could directly inhibit the mRNA expression in Leydig cells for IGF-I, an important autocrine modulator of Leydig cell function. This suggests that the effect of IL-1 beta on Leydig cell function is, at least in part, achieved by down-regulation of IGF-I mRNA levels.     

Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. Patients were intradermally injected with an HLA-A*2402-restricted, natural, or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses as the principal endpoints. Twenty-six patients received one or more WT1 vaccinations, and 18 of the 26 patients completed WT1 vaccination protocol with three or more injections of WT1 peptides. Toxicity consisted only of local erythema at the WT1 vaccine injection sites in patients with breast or lung cancer or acute myeloid leukemia with adequate normal hematopoiesis, whereas severe leukocytopenia occurred in patients with myelodysplastic syndrome with abnormal hematopoiesis derived from WT1-expressing, transformed hematopoietic stem cells. Twelve of the 20 patients for whom the efficacy of WT1 vaccination could be assessed showed clinical responses such as reduction in leukemic blast cells or tumor sizes and/or tumor markers. A clear correlation was observed between an increase in the frequencies of WT1-specific cytotoxic T lymphocytes after WT1 vaccination and clinical responses. It was therefore demonstrated that WT1 vaccination could induce WT1-specific cytotoxic T lymphocytes and result in cancer regression without damage to normal tissues.     

The insulin-like growth factor receptor 1 is a promising target for novel treatment approaches in neuroendocrine gastrointestinal tumours

Gastrointestinal neuroendocrine tumours (NET) represent a heterogeneous tumour entity. The anti-neoplastic therapy of advanced NET disease is still unsatisfactory and innovative therapeutic approaches are needed. As NET frequently express insulin-like growth factors (IGFs) and their receptors (IGFR), known to promote survival, oncogenic transformation, tumour growth and spreading, the inhibition of the IGF/IGF-receptor system may offer possibilities for novel targeted treatment strategies of NET. Here, we studied the anti-neoplastic effects of an inhibition of the IGF-I receptor (IGF-1R) signalling in NET cells by the novel IGF-1R tyrosine kinase (TK) inhibitor NVP-AEW541, whose anti-neoplastic potency has not yet been tested in NET disease. Using two human NET cell lines with different growth characteristics, we demonstrated that NVP-AEW541 dose-dependently inhibited the proliferation of NET cells by inducing apoptosis and cell cycle arrest. Anti-neoplastic effects of NVP-AEW541 were also detected in primary cultures of human neuroendocrine gastrointestinal tumours. Apoptosis was characterized by activation of the apoptotic key enzyme, caspase-3, as well as by detection of changes in the expression of the pro- and anti-apoptotic proteins, BAX and Bcl-2, after NVP-AEW541 treatment. Cell cycle was arrested at the G1/S checkpoint. The anti-neoplastic effects of NVP-AEW541 involved the inactivation of ERK1/2. Induction of immediate cytotoxicity did not account for the anti-neoplastic effects of NVP-AEW541, as shown by measurement of lactate dehydrogenase release. Moreover, additive anti-neoplastic effects were observed when NVP-AEW541 was combined with cytostatics such as doxorubicin or the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin. This is the first report on the induction of apoptosis and cell cycle arrest by the IGF-1R-TK inhibitor, NVP-AEW541, in NET cells. The inhibition of the IGF/IGFR system appears to be a promising novel approach for future treatment strategies of NET disease.     

Octreotide inhibits insulin-like growth factor-I hepatic gene expression in the hypophysectomized rat: evidence for a direct and indirect mechanism of action.

Somatostatin and somatostatin analogs are known to interact with the GH-insulin-like growth factor (IGF)-I axis by inhibiting GH secretion and consequently hepatic IGF-I production. Indirect evidence suggests that octreotide, a somatostatin analog, reduces serum IGF-I levels relatively more than expected from GH reduction, implying a GH-independent pathway of action. To study the role of octreotide in the regulation of IGF-I production, independently of endogenous GH, we used the hypophysectomized (hypox) rat to measure hepatic IGF-I expression and also employed cultured rat hepatocytes to examine whether octreotide has any direct effect on the production of IGF-I. Forty male hypox Sprague-Dawley rats were randomized into 4 groups to receive daily injections for 3 days of either saline, human GH (hGH) (100 g), octreotide (100 g twice), or both hGH (100 g) and octreotide (100 g twice). GH stimulated serum IGF-I levels to 104 +/- 10 micrograms/liter as compared to saline (26 +/- 2 micrograms/liter). Octreotide alone had no effect, but combining octreotide and hGH significantly reduced the hGH-induced rise in the IGF-I levels (52 +/- 6 micrograms/liter). The relative expression of hepatic IGF-I in the rats treated with hGH increased by 4-fold compared to that in the saline-treated rats. Octreotide administered simultaneously with hGH potently blocked the hGH-induced IGF-I expression to control levels. In cultured hepatocytes, IGF-I mRNA levels maximally stimulated by combining bGH and glucagon were significantly inhibited in the presence of octreotide at low concentrations (0.3 and 3 ng/ml) by 25% and 45%, respectively. In contrast, high concentrations of octreotide (30 and 300 ng/ml) had no significant effect on IGF-I mRNA abundance. We conclude that: 1) octreotide inhibits IGF-I serum levels and hepatic gene expression in the hypox rat; and 2) octreotide can inhibit partially the direct effects of GH and glucagon on hepatic IGF-I production.