Transcriptional regulation of insulin-like growth factor-I receptor gene expression in prostate cancer cells.

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5'-flanking sequence, 640 bp of 5'-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5'-flanking region construct was 53% lower (P < 0.0001), and that of the 5'-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor protein for prostate cancer.

Insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1) has been shown to have decreased expression in the progression from benign to malignant prostate epithelial cells (V. Hwa et al., J. Clin Endocrinol. Metab., 83: 4355-4362, 1998). The present study was undertaken to determine the effects of the re-expression of IGFBP-rP1 in a cell line from a model of human prostate cancer, M12, in which IGFBP-rP1 expression had been demonstrated to decrease from the parent epithelial cell, P69, to the malignant subline, M12. An IGFBP-rP1 cDNA encoding the protein was transfected into M12 cells in a plasmid that resulted in constitutive-expression of IGFBP-rP1. Clones of transfected M12 cells were selected for low (L) and high (H) levels of expression, and the plasmid vector alone was transfected into M12 as a control. After transfection, there was a marked alteration in the morphology of the M12 cells such that the H clones had an elongated appearance when compared with the M12 control cells. The M12 clones overexpressing IGFBP-rP1 had a dose-related increase in population doubling time, decreased colony formation in soft agar, an increased propensity to undergo apoptosis in response to 6-hydroxyurea, and decreased tumor formation in male athymic, nude mice. These data suggest that IGFBP-rP1 may have a suppressive effect on prostate cancer development.

Overexpression of an inhibitory insulin-like growth factor binding protein (IGFBP), IGFBP-4, delays onset of prostate tumor formation.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) have been shown to either inhibit or enhance the action of IGF, or act in an IGF-independent manner in the prostate. We have overexpressed the IGF-inhibitory IGFBP-4 in the malignant M12 prostate epithelial cell line to determine the effects on tumor formation and apoptosis. Overexpression was determined by Northern, Western immunoblot and Western radioligand blot analysis. IGF-induced proliferation was reduced in the IGFBP-4 transfected cells compared with control cells (P < or = 0.01). Colony formation in soft agar was significantly inhibited up to 14 days after plating in the IGFBP-4 transfected cells when compared with the M12 controls (P < or = 0.01): however, in the presence of des(1-3)IGF-I, there was no significant difference between the control and IGFBP-4 transfectants in colony formation in soft agar. Apoptosis in an IGFBP-4 transfected cell line was significantly increased in response to induction by 6-hydroxyurea compared with the control line. When injected s.c. into male athymic/nude mice, a marked delay was noted in tumor formation in animals receiving IGFBP-4 transfected cells (P < or = 0.01). Interestingly, IGFBP-2 protein levels were reduced in the conditioned media of all IGFBP-4 transfected cell cultures. These data indicate that an inhibitory IGFBP may significantly delay the growth of malignant prostate epithelial cells and enhance the sensitivity of these cells to apoptosis.

Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: evidence that insulin-like growth factor is required for skeletal muscle differentiation.

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. 125I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1-3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete.

Interleukin-15 stimulates C2 skeletal myoblast differentiation.

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle, and which stimulates muscle protein accretion in cultured skeletal muscle fibers. Using parental C2 skeletal myoblasts, no significant effects of IL-15 on skeletal muscle differentiation were observed. To test the hypothesis that IL-15 may stimulate skeletal muscle differentiation if the strong differentiation-inducing effects of autocrine insulin-like growth factor (IGF) production were inhibited, a C2 myoblast subline (C2-pBP4) was stably transfected with an expression vector for rat IGF binding protein-4 (IGFBP-4). Differentiation responses to autocrine and exogenous IGFs in C2-BP4 myoblasts were reduced 3- to 4-fold in C2-BP4 cultures compared to C2-pLXSN cultures, a subline transfected with a control plasmid. Addition of IL-15 to C2-pBP4 myoblasts doubled the number of differentiated muscle cells which arose. These findings indicate that IL-15 can stimulate myogenic differentiation in conditions in which the strongly differentiative effects of the IGFs are inhibited. The differentiative activity of IL-15 may be of physiological significance in conditions in which IGF concentrations are low or in which the IGFs are sequestered by binding proteins.

Developmental regulation of Mac25/insulin-like growth factor-binding protein-7 expression in skeletal myogenesis.

Mac25 is a newly discovered member of the insulin-like growth factor-binding protein (IGFBP) family, recently assigned the name IGFBP-7, Mac25/IGFBP-7 is hypothesized to have growth-suppressing activity, since mac25/IGFBP-7 mRNA is down-regulated in several tumor cell lines and is highly expressed in senescent mammary epithelial cells. In this study, mac25/ IGFBP-7 mRNA expression was characterized in the C2 skeletal myogenic cell line, which undergoes a transition from actively dividing, undifferentiated myoblasts to nondividing, differentiated myotubes. Mac25/ IGFBP-7 mRNA levels were 2.5-fold higher in dividing C2 myoblasts than in nondividing myotubes. The inverse correlation between mac25/IGFBP-7 expression and myogenic differentiation was further examined by treating myogenic cultures with transforming growth factor-beta (TGF-beta) or insulin-like growth factor-I (IGF-I). TGF-beta inhibited myogenic differentiation by 98% and stimulated mac25/IGFBP-7 mRNA expression 2-fold. IGF-I stimulated differentiation by 50% and inhibited mac25/IGFBP-7 expression 2- to 3-fold. These findings indicate that, in contrast to other cell systems examined so far, expression of this new member of the IGFBP family is not always correlated with a nonproliferative state.

Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv Bartlett.

A polygalacturonase inhibitor glycoprotein with an apparent molecular mass of 43 kD was purified from pear (Pyrus communis L. cv Bartlett) fruit. Chemical deglycosylation of this protein decreased the molecular mass to 34 kD. Gas chromatographic analysis suggests that N-linked glycosylation accounts for the majority of sugar moieties. Partial amino acid sequence analysis of the purified polygalacturonase inhibitor protein provided information used to amplify a corresponding cDNA by polymerase chain reactions. Multiple cloned products of these reactions were sequenced and the same open reading frame was identified in all of the products. It encodes a 36.5-kD polypeptide containing the amino acid sequences determined by protein sequencing and predicts a putative signal sequence of 24 amino acids and seven potential N-glycosylation sites. The expression of polygalacturonase inhibitor is regulated in a tissue-specific manner. Activity and mRNA level were much higher in fruit than in flowers or leaves.