Positive Growth Regulators Research Articles

Molecular karyotype (amplotype) of metastatic colorectal cancer by unbiased arbitrarily primed PCR DNA fingerprinting.

Genomic instability characterizes the aneuploid cancer cell. Losses of genetic material are critical in cancer by exposing recessive mutations in tumor suppressor genes. Gains of genetic material also may lead to overexpression of genes contributing to tumor progression either in the presence or absence of mutation. However, the detection of moderate gains (such as tri-tetraploidy) has been a challenge in cancer research. Unbiased DNA fingerprinting by the arbitrarily primed PCR allows the detection of moderate gains (in addition to losses) of DNA sequences of known chromosomal localization. We have generated in this manner a molecular karyotype of metastatic colon cancer. This amplotype shows that sequences from several chromosomes undergo both losses (1, 4, 9, 14, and 18) and gains (6, 7, 12, and 20) in over half of the tumors. Moreover, gains of sequences from chromosomes 8 and 13 occurred in most tumors, indicating the existence in these chromosomes of positive regulators of cell growth or survival that are under strong positive selection during tumor progression. We conclude that overrepresentation of these chromosomal regions is a critical step for metastatic colorectal cancer. Comparative amplotype analysis from primary and metastatic tumors suggest the existence in chromosome 4 of gene(s) whose loss is specifically selected in cells that reach the metastatic stage.

Regulation of cyclin G1 during murine hepatic regeneration following Dipin-induced DNA damage.

Cyclin G1 has been linked to both positive and negative growth regulation. The expression of cyclin G1 is induced by transforming growth factor beta1 and p53, as well as by multiple mitogenic stimuli in mammalian cells in culture. However, the physiological role of cyclin G1 remains unclear. To examine the cell-cycle regulation of cyclin G1 in vivo, two models of coordinated cell proliferation induced by partial hepatectomy (PH) in the presence or absence of DNA damage were used. To introduce DNA damage, mice were treated with the alkylating drug, 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin) 2 hours before PH. Cell-cycle progression was monitored by 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA, the frequency of mitoses, the expression of cell-cycle control genes, and by flow cytometry. Dipin treatment resulted in cell-cycle arrest at the G2/M boundary without affecting G0/G1 and G1/S transitions. While the hepatocytes progressively entered G2 phase arrest, the cyclin G1 mRNA and protein levels increased more than five- and eightfold, respectively. Cyclin G1 had a nuclear localization in all interphase cells with clear absence from nucleoli. In contrast, during mitosis, cyclin G1 was undetectable by immunohistochemistry. Taken together, our data provide evidence for a putative role of cyclin G1 in G2/M checkpoint control.

Characteristics of Smooth Muscle Cell Lipoprotein Binding Proteins (p105/p130) as T-Cadherin and Regulation by Positive and Negative Growth Regulators

Smooth muscle cells (SMC) express atypical surface low density lipoprotein (LDL) binding proteins of Mr105 and Mr130 (p105 and p130) which have been putatively identified as the cell adhesion glycoprotein T-cadherin. Using cultured human and rat aortic SMC and analysis by ligand (LDL)- and immuno-blotting techniques we now confirm identity of p105 and p130 as T-cadherin, as adjudged by sensitivity to PI-PLC cleavage, insensitivity to trypsin degradation in the presence of calcium, and immunoreactivity to anti-T-cadherin peptide antisera. The function of T-cadherin (p105/p130) in the vasculature is unknown. The proteins were downmodulated by the peptide growth factors PDGF-BB, IGF, EGF, and bFGF, but not by vasoactive peptide hormones (angiotensin II, vasopressin, bradykinin, and endothelin). TGFβ, a recognized inhibitor of SMC proliferation, per se had no effect but inhibited growth factor-induced p105/p130 downmodulation. Expression of p105/p130 in quiescent SMC and growth-stimulated SMC (respectively, in serum-free and serum or PDGF-BB containing culture conditions) was increased by forskolin and 8-Br-cyclic GMP, both anti-mitogenic substances, but was unaffected by phorbol ester, calcium ionophores, or calcium antagonists. The findings are compatible with a function for the lipoprotein binding proteins (T-cadherin) in negative regulation of SMC growth.

Identification of a Novel AP-2 Consensus DNA Binding Site

Activator Protein (AP)-2 is a transcription factor that is required for mouse development. AP-2 activates expression of positive and negative growth regulators including erbB-2 and p21WAF1/CIP1. Induction of p21 has been correlated with cell cycle and growth inhibition of human cancer cells. Because several endogenous AP-2 binding sites do not fit the known consensus sequences well, we sought to define AP-2's interaction with DNA more precisely. UsingCyclicAmplification andSelection ofTargets (CAST'ing) of random oligonucleotide sequences and recombinant human AP-2 protein, we identified 17 novel AP-2 binding sites. Mobility shift assays showed significant AP-2 binding of the novel sites as compared to p21, erbB-2 and hMtIIa sites. Several sites that bound with high specificity and affinity did not fit known AP-2 consensus sequences. A sequence comparison based on several of the novel sequences yielded a putative consensus binding sequence of 5′-TAGAAAGNYCYNG-3′. These DNA binding sites may help identify novel targets of AP-2 and aid in further understanding AP-2 function.

Effect of interleukin-1 and dexamethasone on interleukin-6 production and growth in human meningiomas.

Interleukin-6 (IL-6) has been shown to be released by cultured human meningioma cells and may be a positive or negative regulator of tumour growth. IL-6 protein and mRNA levels have been examined in a series of meningiomas. In 14 cases, the results are compared with the effects of IL-6 and dexamethasone on growth and IL-6 secretion in vitro. Tumours with the highest in vivo IL-6 mRNA expression also showed maximum induction of IL-6 and increased cellular proliferation on IL-1 stimulation in vitro. Dexamethasone decreased the IL-1-stimulated IL-6 release in all cases. Meningiomas which had little or no IL-6 message were refractory to IL-1 control of IL-6. Remarkably, these formed the group of meningiomas that increased their growth rate in response to dexamethasone.

Effects of interleukin-1 and dexamethasone on interleukin-6 production and growth in human meningiomas

Interleukin-6 (IL-6) has been shown to be released by cultured human meningioma cells and may be a positive or negative regulator of tumour growth. IL-6 protein and mRNA levels have been examined in a series of meningiomas. In 14 cases, the results are compared with the effects of IL-6 and dexamethasone on growth and IL-6 secretion in vitro. Tumours with the highest in vivo IL-6 mRNA expression also showed maximum induction of IL-6 and increased cellular proliferation on IL-1 stimulation in vitro. Dexamethasone decreased the IL-1-stimulated IL-6 release in all cases. Meningiomas which had little or no IL-6 message were refractory to IL-1 control of IL-6. Remarkably, these formed the group of meningiomas that increased their growth rate in response to dexamethasone. © 1997 John Wiley & Sons, Ltd.

Conserved and novel regulators of the plant cell cycle

Cell division is highly regulated, both spatially and temporally, during plant development. Recent evidence implicates cyclin-dependent kinases (cdks) and their associated proteins as the principal temporal regulators of cell division. It is now known that plants contain an extended family of cdks, some of which appear to be unique to this group. Positive rate-limiting regulators of cell proliferation and growth include mitotic or B-type cyclins whose transcription is restricted to the G 2 and M phases. Current research suggests that MYB-related transcription factors may be responsible for this restriction. Cdk-interacting proteins, such as cdk inhibitors and suc1 homologues, have been isolated using yeast two-hybrid approaches.

Molecular mechanisms involved in the nutritional and hormonal regulation of growth in pigs.

Nutritional and hormonal factors are major determinants of animal growth and, postnatally at least, pituitary-derived growth hormone (GH), via its stimulatory effects on insulin-like growth factor-I (IGF-I) production, is one of the major regulators of growth. The production of GH by the pituitary is under the local control of growth-hormone-releasing factor (GRF) and growth-hormone-release-inhibiting factor (somatostatin). However, diet also has a major effect on circulating GH concentrations, such that nutritional restriction leading to growth arrest is associated with a decrease in plasma IGF-I concentration, but an increase in plasma GH concentration in most species, including the pig (Buonomo & Baile, 1991), the rat being the exception. The molecular mechanisms for this growth arrest, in the presence of high concentrations of this positive regulator of growth (GH), are proposed to be via dietary effects on the components of the GH-IGF axis, and in particular IGF-I, and form the subject for the present review.

Transforming growth factors alpha & beta — Positive and negative regulators of epithelial growth

Transforming growth factors alpha & beta — Positive and negative regulators of epithelial growth

Proteolytic Activation of Rim1p, a Positive Regulator of Yeast Sporulation and Invasive Growth

In the yeast Saccharomyces cerevisiae, rim1, 8, 9, or 13 mutations cause four phenotypes: poor growth at low temperature, altered colony morphology, inefficient sporulation due to reduced expression of the meiotic activator IME1, and, as shown here, defective invasive growth. In this report, we have determined the relationship between RIM1 and the other genes, RIM8, 9, and 13, in this group. We have analyzed production of epitope-tagged Rim1p derivatives with HA epitopes at the N-terminus or in the middle of the protein. These Rim1p derivatives exist primarily as a small form (90 kD for Rim1-HA2p) in wild-type cells and as a large form (98 kD for Rim1-HA2p) in rim8, 9, and 13 mutants. We have also analyzed production of beta-galactosidase in strains that express a RIM1-lacZ fusion gene. beta-galactosidase exists primarily as a approximately 130 kD form in wild-type cells and as a approximately 190 kD form in rim9 mutants. These results indicate that Rim1p undergoes C-terminal proteolytic cleavage, and that rim8, 9, and 13 mutations block cleavage. Expression of a Rim1p C-terminal deletion derivative suppresses rim8, 9, and 13 mutations. Thus the phenotypes of rim8, 9, and 13 mutants arise from the defect in Rim1p C-terminal cleavage. Cleavage of Rim1p, like that of its Aspergillus nidulans homologue PacC, is stimulated under alkaline growth conditions. Therefore, Rim1p, PacC and their respective processing pathways may represent a conserved signal transduction pathway.

Clinical significance of the determination of angiogenic factors

INTRODUCTION ANGIOGENIC ACTIVITY is known to depend upon a balance between positive and negative angiogenesis regulators [ 11. The alteration of this balance by mechanisms, such as the induction of vascular endothelial growth factor (VEGF), expression of TP53 or RAS mutations, SRC overexpression, and downregulation of thrombospondin-1 expression by TP53 mutation, can switch the angiogenic phenotype and facilitate the growth of solid tumours [2-51. Currently, measurement of intratumoral microvessel density (IMD) by immunocytochemistry appears to be the most reliable method of measuring angiogenic activity [6]. However, the determination of levels of each endothelial growth regulator in tumour tissues and in serum or urine can provide more information on the angiogenic characteristics of each tumour and might be an alternative method for evaluating angiogenic activity. For example, the level of intratumoral VEGF varies greatly between tumours and can be a valuable prognostic factor [7]. The endogenous angiogenesis inhibitor, angiostatin, which can cause tumour regression in human tumour xenografts models, has been detected in serum [8]. Since the biological properties of each endothelial growth factors is different, it will be of particular importance to know the role of each factor in human tumours. Therefore, we have focused on the determination of angiogenic factors, particularly positive endothelial growth regulators including VEGF, platelet-derived endothelial cell growth factorithymidine phosphorylase (I’D-ECGFITP), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and metalloproteinases (MM&), in breast cancer patients. In this article, we review our experience and the most relevant data in the literature on the clinical significance of the determination of these endothelial growth regulators in human tumours.

Tumor Necrosis Factor-α-Induced Apoptosis in a Human Keratinocyte Cell Line (HaCaT) Is Counteracted by Transforming Growth Factor-α

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-α (TGF-α) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-α (TNF-α) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-α induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation (“DNA laddering”), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-α. The protective effect of TGF-α was abrogated by translation inhibition, indicating that it depended onde novoprotein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-α-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-α.

Transforming Growth Factor-α in Carcinogen- Induced F344 Rat Hepatic Foci

Transforming growth factor-α (TGFα) is a positive growth regulator in epithelial cells, including hepatocytes. Overexpression of TGFα has been associated with increased growth and malignancy of end-stage cancers in humans and rodents. The overall aim of this study was to characterize TGFα staining in diethylnitrosamine-induced hepatic foci from male F344 rats with the hematoxylin and eosin (H and E) histological phenotype. The association between the individual focal DNA synthesis labeling index and the presence of TGFα was also examined. Hepatic foci were identified as eosinophilic, basophilic, clear cell, or mixed cell. Of these foci, 37.5% labeled positive for TGFα. There were distinct differences in the pattern of TGFα labeling between the different H and E histological phenotypes. Intense, uniform TGFα labeling was observed in eosinophilic foci. Basophilic foci labeled for TGFα diffusely uniform throughout the cytoplasm. In clear-cell foci, TGFα labeling occurred primarily along the periphery of the cell membrane. In mixed-cell foci, labeling occurred both along the periphery and diffusely throughout the cytoplasm. On those slides stained, glutathione-S-transferase (placental; GSTP) was detected in almost all eosinophilic and mixed-cell foci, whereas approximately half of the basophilic and clear-cell foci stained for GSTP. The presence of GSTP in a focus was not always associated with the presence of increased TGFα protein. All rat hepatic adenomas and the one carcinoma labeled positive for TGFα. Increased levels of TGFα protein were associated with increased DNA synthesis labeling index. The number of TGFα-positive foci with the highest DNA synthesis labeling indices were statistically higher than those with lower levels of DNA synthesis labeling. Although characteristic staining patterns for TGFα were associated with specific histological subtype, the role that TGFα plays in the progression of focal lesions to neoplasia requires further definition. High levels of TGFα protein appear to be acquired sometime during the hepatocarcinogenic process. It may be that early lesions that acquire high levels of TGFα are the ones to develop into hepatocellular carcinoma (e.g., hepatocellular carcinoma is determined very early in the carcinogenic process). It is apparent that further work is needed to delineate the role of TGFα in both rodent and human hepatocarcinogenesis.

Regulation of prostatic growth and function by peptide growth factors.

Polypeptide growth factors are positive and negative regulators of prostatic growth and function. Expression and biological effects of epidermal growth factor (EGF), transforming growth factors (TGFs) alpha and beta, fibroblast growth factors (FGFs), and insulin-like growth factors (IGFs) in the prostate have been extensively studied. EGF and TGF alpha, which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells. Their paracrine mode of action in normal tissue and early-stage tumors is apparently altered towards an autocrine stimulation in hormone-independent tumors, which gain the ability to produce TGF alpha by themselves. TGF beta has a dual role in the regulation of prostatic growth. It inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to the inhibitory effect of TGF beta. Several members of the FGF family have been identified in the prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF (bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether prostatic cancer cells gain the ability to produce IGF-I by themselves and thus shift to an autocrine mode of IGF-I stimulation. Growth factors also interact with the androgen-signaling pathway. IGF-I in particular, other growth factors as well, can activate the androgen receptor.

Sphingoid bases and phospholipase D activation

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine- l-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.

Differential expression of novH and CTGF in human glioma cell lines

Aims-(1) To investigate the expression in human derived glioblastoma cell lines of two structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), which encode putative insulin-like growth factor binding proteins of a novel type. (2) To investigate whether the same transcription factors regulate CTGF and novH expression.Methods-Expression of novH and CTGF was analysed in 24 glioblastoma derived cell lines by northern blotting. The CTGF promoter region was characterised by nucleotide sequencing, RNase protection experiments, by transient transfections, and CAT assays.Results-CTGF and novH mRNA levels differed in the glioma cell lines studied. NovH and CTGF genes were not co-expressed in all cell lines. The CTGF promoter region was highly conserved compared with the corresponding region in the mouse (FISP12) and exhibited in vitro transcriptional activity.Conclusions-Although the coding regions of novH and CTGF are highly homologous, their promoter regions are substantially different, suggesting that these two genes may be regulated by different mechanisms. Considering that novH and CTGF are likely to be, respectively, negative and positive regulators of growth and that some glioma cell lines expressing novH are not tumorigenic, expression of these two genes might represent a key element in determining the stage of differentiation or the malignant potential, or both, of some tumour cell lines.

Requirement of Transforming Growth Factor-β (TGF-β) Type II Receptor for TGF-β-induced Proliferation and Growth Inhibition

Growth regulation of fibroblasts is important for lung development and repair of lung injury. In this study, we investigated the role of transforming growth factor-beta (TGF-beta) type II receptor in the TGF-beta-dependent proliferative response of lung fibroblasts. TGF-beta stimulated the proliferation of adult lung fibroblasts at a low concentration (1 ng/ml), but inhibited the growth of fetal lung fibroblasts in a dose-dependent fashion (0.1-10 ng/ml). Cross-linking and Northern analysis demonstrated that the two lung fibroblast cell lines expressed the TGF-beta type I receptor (T beta RI) and type II receptor (T beta RII). We overexpressed in lung fibroblasts a truncated derivative of T beta RII that lacked the cytoplasmic serine/threonine kinase domain (T beta RII delta K). T beta RII delta K was a dominant-negative inhibitor of TGF-beta signal transduction blocking not only TGF-beta-induced mitogenic action upon adult lung fibroblasts but also TGF-beta-induced growth inhibition of fetal lung fibroblasts. The results indicate that the type II receptor is indispensable for mediating both the mitogenic and antiproliferative effects of TGF-beta upon lung fibroblasts.

Role of c-myc in myeloid differentiation, growth arrest and apoptosis.

Hematopoiesis is a profound example of cell homeostasis which is regulated throughout life, whereby a hierarchy of hematopoietic progenitor cells in the bone marrow (BM) proliferate and terminally differentiate along multiple, distinct cell lineages. This includes the proliferation and differentiation of myeloid precursor cells into granulocytes and macrophages (33, 40, 42). Clearly, a variety of control mechanisms are needed to maintain steady state levels of mature blood cells, as well as to stimulate the rapid production of specific cell types as needed. To achieve this requires the participation of many factors, including positive and negative regulators of growth and differentiation, which determine survival, growth stimulation, differentiation, functional activation, and programmed cell death (apoptosis) (41, 44).

Differential regulation of c-Jun and JunD by ubiquitin-dependent protein degradation.

c-Jun and JunD are two closely related members of the Jun family of transcription factors which markedly differ in their biological functions. Whereas c-Jun behaves as a positive regulator of cell growth and may cause cell transformation when overexpressed, JunD antagonizes both of these effects. To better understand how the activities of c-Jun and JunD are controlled, we investigated how their stabilities within the cell are determined. We show that, in contrast to c-Jun which is degraded following multi ubiquitination, JunD is not efficiently ubiquitinated and exhibits a correspondingly longer half-life. Mutational analysis reveals that the determinant for the difference in ubiquitination resides in the NH2-terminal regions of the proteins which in c-Jun contains the delta-domain.

Differential effects of glycosaminoglycans on neurite growth on laminin and L1 substrates

Glycosaminoglycans (GAGs), the carbohydrate moieties of proteoglycans, are thought to be positive and negative regulators of axonal growth. The physiological role of GAGs is controversial as some studies have shown that GAGs inhibit cell adhesion and neurite elongation (Exp Neurol 109:111, 1990) whereas other studies have reported a growth stimulatory effect of GAGs (Development 114:17, 1992). These and other studies have examined the effects of GAGs using different types of neurons and different substrate conditions thereby making a direct comparison of the experimental data difficult. To resolve the controversy concerning the ability of exogenous GAGs to modulate neurite growth, we examined the effects of a panel of structurally different GAGs on the growth of postnatal rat cerebellar granule neurons and embryonic rat dorsal root ganglia (DRG) neurons on substrates of either laminin or the L1 glycoprotein. Here we show that chondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), and keratan sulfate (KS) inhibit neurite growth from both cerebellar and DRG neurons on laminin-coated surfaces. On L1 surfaces, however, these GAGs are either extremely weak inhibitors of neurite extension or, in the case of CS4, a modest stimulator of neurite growth. Heparan sulfate (HS) and dermatan sulfate (DS) inhibited the growth of cerebellar neurons but not the growth of DRG neurons on L1-coated surfaces. On laminin surfaces, DS and HS had no effect on neurite growth from both cerebellar and DRG neurons. These results demonstrate a cellular and a substrate specificity to the effects of exogenous GAGs on neurite extension in vitro. They suggest that while CS and KS GAGs may not exert strong negative influences over axonal growth in regions of the developing CNS where the L1 glycoprotein is abundant, these GAGs are capable of inhibiting the growth of axons that extend within an environment rich in laminin.