Regulation Of Normal Growth Research Articles

A functional analysis of imprinting in parthenogenetic embryonic stem cells

A detailed analysis of the developmental potential of parthenogenetic embryonic stem cells (PGES) was made in vivo and in vitro, and a comparison was made with the development of cells from parthenogenetic embryos (PG). In vivo, in chimeras with normal host cells (N), PGES cells showed a restricted tissue distribution consistent with that of PG cells, suggesting faithful imprinting in PGES cells with respect to genes involved in lineage allocation and differentiation. Restricted developmental potential was also observed in teratomas formed by ectopic transfer under the kidney capsule. In contrast, the classic phenotype of growth retardation normally observed in PG<==>N chimeras was not seen, suggesting aberrant regulation in PGES cells of genes involved in growth regulation. We also analysed the expression of known imprinted genes after ES cell differentiation. Igf2, H19 and Igf2r were all appropriately expressed in the PGES derived cells following induction of differentiation in vitro with all-trans retinoic acid or DMSO, when compared with control (D3) and androgenetic ES cells (AGES). Interestingly, H19 was found to be expressed at high levels following differentiation of the AGES cells. Due to the unexpected normal growth regulation of PGES<==>N chimeras we also examined Igf2 expression in PGES derived cells differentiated in vivo and found that this gene was still repressed. Our studies show that PGES cells provide a valuable in vitro model system to study the effects of imprinting on cell differentiation and they also provide invaluable material for extensive molecular studies on imprinted genes. In addition, the aberrant growth phenotype observed in chimeras has implications for mechanisms that regulate the somatic establishment and maintenance of some imprints. This is of particular interest as aberrant imprinting has recently been invoked in the etiology of some human diseases.

Overexpression of the protein tyrosine phosphatase PTP1B in human breast cancer: association with p185c-erbB-2 protein expression.

The p185c-erbB-2 growth factor receptor protein tyrosine kinase (PTK) is overexpressed in one third of human breast cancer patients and indicates a poor prognosis in these patients. Protein tyrosine phosphatases (PTPs) may balance PTK activity as part of normal growth-regulation pathways. PTP1B is an intracellular PTP that is involved in linkage between signal transduction pathways and may interface with inappropriate PTK activity in transformed cells. The aim of this study was to determine if PTP1B is overexpressed in human mammary tumors and to determine if such overexpression is associated with the overexpression of the p185c-erbB-2 receptor PTK. Our samples were frozen sections from 29 human mammary tumors (19 pure infiltrating, two pure intraductal, and eight combined intraductal and infiltrating) and nine sections from normal breast tissue. The sections were immunohistochemically stained for PTP1B and p185c-erbB-2, and the results were analyzed statistically for association between overexpression of the two proteins. Northern blot analysis was used to assess if PTP1B overexpression was coincident with increased transcription of the PTP1B gene. Overexpression of the PTP1B protein was observed in 72.4% of the tumor sections compared with normal epithelium, with maximal expression occurring in 37.9% of the tumors. All of the tumor subtypes, including the pure intraductal lesions, overexpressed PTP1B. Statistical analyses demonstrated a significant association between PTP1B overexpression and breast cancer (P < .038) and between the overexpression of PTP1B and the overexpression of p185c-erbB-2 (P < .006). PTP1B messenger RNA steady-state transcription was consistently increased in tumors versus normal tissues. PTP1B overexpression is a common phenotypic manifestation in human breast cancers and is associated with over-expression of p185c-erbB-2. Steady-state PTP1B transcription is increased in tumor tissue. Further studies are needed to determine if PTP1B overexpression balances or augments PTK activity. PTP1B overexpression might also be evaluated as a clinical prognostic factor in human breast cancers.

Dual specificity kinases--a new family of signal transducers.

Phosphorylation/dephosphorylation reactions are one of the dynamic mechanisms through which cells modulate protein activity in response to environmental stimuli. The eukaryotic molecules which are responsible for the phosphorylation of serine, threonine and tyrosine residues appear to have co-ordinately evolved from simple prokaryotic enzymes which primarily respond to nutritional cues. In multicellular eukaryotes the complexity of data transfer greatly exceeds that of simple bacteria. The eukaryotic cell needs to exchange information with neighbouring and distant sister cells. Positional, nutritional and hormonal data are transmitted from the extracellular milieu across the plasma membrane and into the cytoplasm. In certain cases the signal must pass into the nucleus or other subcellular organelles where it is decoded and the proper cellular response initiated. All of these events have been shown to have a protein kinase component and it seems likely that in mammalian cells over 1,000 different kinase molecules have evolved to form the requisite signal transducing networks. In this review we describe a previously unappreciated family of protein kinases, the dual specificity or DSK kinases, which play important roles in the regulation of normal cellular growth and differentiation.

Autoantibodies to Hair Follicles in Normal Individuals

Indirect evidence suggests that immune mechanisms may be involved in the pathogenesis of some hair disorders, eg, alopecia areata, 1 and in the regulation of normal hair growth. 2 However, no direct evidence of an immune-response selectively directed to hair follicle (HF) antigens, an essential requirement if these hypotheses are correct, has been reported hitherto. In this study, we report the presence of autoantibodies to HFs in normal individuals. Methods. Serum samples from 22 normal individuals (four autologous and 18 allogeneic) were tested by Western immunoblotting for antibodies to anagen HFs from four normal individuals. Anagen hair follicles were plucked from normal scalp, identified on the basis of their prominent outer root sheath and bulb, and collected. Control tissues included scalp epidermis and dermis obtained by surgical procedure and cultured normal epidermal keratinocytes. Proteins were extracted with 6 mol/L of urea and separated by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis,

Changes in UsnRNA biosynthesis during rat liver regeneration.

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in UsnRNA biosynthesis during rat liver regeneration.

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30-50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the deleted in colorectal cancer gene in normal and neoplastic tissues.

Chromosome 18q is among the chromosomal regions thought to harbor a tumor suppressor gene(s) that is frequently inactivated during the development of several cancer types, particularly those of the gastrointestinal tract. Moreover, preliminary data suggest that colorectal cancers with 18q LOH have a more aggressive clinical behavior than those cancers without 18q LOH. A candidate tumor suppressor gene from 18q, termed DCC for deleted in colorectal cancer, has been identified. The DCC gene is contained within the common region of LOH on 18q, its expression is markedly decreased or absent in colorectal cancers and cell lines, and a subset of colorectal cancers have been shown to have somatic mutations within the DCC gene. Thus, DCC represents the most promising candidate tumor suppressor gene from 18q. At present, however, many questions remain regarding the mechanisms underlying the inactivation of DCC in the majority of colorectal cancers. In addition, although studies of 18q LOH and DCC gene expression in other cancer types suggest that DCC inactivation may contribute to the pathogenesis of other tumor types, few studies have provided definitive data to demonstrate that DCC inactivation is a critical genetic event in these tumors. Moreover, little is known about the function of DCC in the regulation of normal cell growth and tumor suppression. The predicted structural similarity of DCC to the N-CAM family of cell-surface proteins suggests that it may function through cell-cell and/or cell-extracellular matrix interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetics and oncogenes in leukemia

Analysis of chromosomal abnormalities present in the malignant cells of patients with leukemia has provided much information of diagnostic and prognostic importance. The cloning of genes rearranged by translocation breakpoints has provided insights into both the regulation of normal cell growth and differentiation and the pathogenesis of human malignancies. This article reviews recent analyses of the association of specific karyotypic changes with other disease characteristics, including treatment outcome. Also covered is the substantial progress that has been made in the molecular analysis of genetic abnormalities in leukemia, with emphasis on those changes produced by chromosomal rearrangements. The use of information derived from these latter studies to develop polymerase chain reaction and fluorescent in situ hybridization assays for clinical use is also summarized.

Viruses and human cancer.

Epstein-Barr virus (EBV), hepatitis B virus (HBV), HTLV-I and some human papillomaviruses (HPVs) appear to contribute to the development of a large proportion of certain human cancers. Although the epidemiological evidence linking infection with these viruses to malignancies is generally convincing, only for the HPVs have experimental systems indicated a clear role for some of the HPV encoded genes in tumour cell growth. In these cases, molecular analysis is revealing the mechanisms by which the virally encoded oncogenes function to perturb the normal regulation of cell growth. For HTLV-I, EBV and HBV the mechanism by which the viruses contribute to tumour cell growth is obscure, even though much has been learned about cell transformation in culture by these viruses. A detailed understanding of the mechanism of oncogenesis will be required to design therapeutic drugs for the treatment of these cancers. Prophylactic vaccination resulting in prevention of infection may be an effective approach to reduce the incidence of some of these common cancers.

Induction of reversible growth retardation and growth hormone deficiency by blockade of norepinephrine synthesis in the rat

Norepinephrine is a major regulator of the release of growth hormone. Diethyldithiocarbamate, a dopamine-beta-hydroxylase inhibitor, reduces norepinephrine synthesis and acutely inhibits growth hormone (GH) secretion. To investigate the long-term effects of dopamine-beta-hydroxylase blockade on growth, we administered diethyldithiocarbamate (0, 40, 100 or 400 mg/kg sc b.i.d.) to 21-day-old female rats for 10 days. Food intake, body weight, and tail length were measured twice a week. Plasma GH levels and hypothalamic dopamine and norepinephrine content were measured; messenger ribonucleic acids (mRNAs) for GH-releasing hormone and somatostatin were determined by quantitative in situ hybridization. Diethyldithiocarbamate administration decreased GH levels (p < 0.05) and retarded growth in a dose-dependent manner (p < 0.05), without altering food intake. Co-administration of GH partially reversed the growth retardation in diethyldithiocarbamate-treated animals (p < 0.05). Diethyldithiocarbamate treatment also increased the hypothalamic dopamine/norepinephrine ratio (1.13 vs 0.41 control, p < 0.05). Local levels of GH-releasing hormone and somatostatin mRNA were not altered by treatment. After discontinuation of diethyldithiocarbamate, growth rates returned to normal or transiently even to supranormal values. Norepinephrine synthesis blockade with diethyldithiocarbamate provides a model for reversible growth retardation, in which GH levels are decreased in the absence of decreased GH-releasing hormone mRNA. These results support a role for norepinephrine in the regulation of normal growth.

ErbB-3, a third member of the erbB/epidermal growth factor receptor gene family: its expression in head and neck cancer cell lines.

Proto-oncogenes encoding growth factor receptors constitute several distinct families with close overall structural homology. The highest degree of homology can be observed in their catalytic domains, which are essential for intrinsic tyrosine kinase activity. Growth factor receptors in several of these families play critical roles in the regulation of normal cell growth and development. Some of these molecules have been implicated in the neoplastic process as well. A related DNA fragment distinct from epidermal growth factor receptor and erbB-2 genes was detected by reduced stringency hybridization of v-erbB to normal genomic human DNA. The expression of erbB-3 was studied by southern and northern blot technique in a subset of nine head and neck tumor cell lines, as well as in three immortalized cultures established from normal human salivary glands. No gene amplification of erb-B-3 was noted in any of the head and neck cell lines. The 6.2 kb transcript of erbB-3 was elevated significantly in an epidermoid carcinoma of the larynx (A388) and an esophageal carcinoma (HA 114).

Mechanisms of reversion.

Reversion of transformed cells provides biological system useful in multiple areas of cancer research. First, reversion can be induced by inhibiting the activities of specific oncogenes or replacing intact tumor suppressor genes, and such experiments yield important insights into the roles of these genes in carcinogenesis and normal growth regulation as well as the functional relationships between these genes and other known genes. Second, novel genes involved in growth regulation can be discovered by isolating revertants from transformed cells after mutagenesis or DNA transfection and then characterizing the genes responsible for the non-transformed phenotype. Third, potential anti-cancer drugs can be screened by reversion assay using cells transformed by specific oncogenes. The novel genes and chemicals discovered through these studies serve as valuable tools that help our understanding of how cells regulate their growth and differentiation.

Peripheral nerve regeneration: Role of growth factors and their receptors

Growth factors play a central role in the regulation of normal and injury-induced regenerative cell growth. The purpose of this article is to summarize the available data on the expression of different growth factors and their receptors in the injured peripheral nervous system and to discuss their possible role in promoting peripheral nerve regeneration.

B-Lymphoma Models for Tolerance: The Good, the Bad, and the Apoptotic

Murine B-cell lymphomas have become useful models for analyzing the mechanisms of clonal deletion and apoptosis during tolerance, as well as for understanding the regulation of both normal and neoplastic cell growth. In this article, the advantages and disadvantages of these lymphoma models are summarized, and the pathways of signal transduction regulating cell cycle behavior are described. Our studies, and those of several other laboratories, have demonstrated that crosslinking of membrane IgM on a subset of B-cell lymphomas leads to an initial tyrosine phosphorylation event leading to the transcriptional regulation of the c-myc oncogene, subsequent modulation of the phosphorylation of the pRB growth suppressor protein, and cell cycle arrest. This process is followed by apoptosis presumably due to alterations in myc protein turnover. Whether similar events occur during B-cell tolerance in the developing host is discussed.

Growth factors: Regulation of normal and neoplastic growth

This paper presents an overview of current trends in growth factor research. The first part of the review considers the current classification of growth factors and their receptors. A model of cell proliferation regulation by growth factors is then presented. The final section reviews the latest concepts of the involvement of growth factors in the development of neoplasia.

In search of the functions of normal p53 protein

p53 is of major importance in protecting cells from neoplasia. Most human tumours have an abnormal or inactivated p53 protein. Furthermore, when expression of normal p53 is reinstated in cancer cell lines, they undergo growth suppression leading to either cell differentiation or programmed cell death. But is p53 involved in the regulation of normal cell growth and differentiation in vivo? Here, Varda Rotter and colleagues critically assess some recent conflicting data on this issue.

Gap-junction protein gene suppresses tumorigenicity.

Prompted by the notion that the membrane channels in gap junctions conduct growth-regulating signals from cell to cell, we transferred the alpha 1 gene for the channel protein (connexin43) of rat heart to tumorigenic mouse MCA-10 cells. Upon incorporation into the cell genome, this exogenous gene was expressed, resulting in functional channels and normal growth regulation: cell-cell communication, determined with a channel-permeant 400-dalton fluorescent tracer, was increased and tumorigenicity, determined in nude mice, was suppressed.

Identification and Characterization of Novel Substrates for Protein Tyrosine Kinases

Publisher Summary This chapter discusses: (1) the strategies used to identify Protein tyrosine kinase (PTK) substrates and (2) the recent progress made in identifying new and interesting cytoskeletal protein components that serve as substrates for oncogenic PTKs. PTKS play an important role in the transmission of signals for a broad spectrum of cellular activities, including the activation of secretory events, T-cell and B-cell activation, responses to cell-extracellular matrix interactions, differentiation, mitogenesis, and oncogenesis. Because of the desire to understand the molecular basis of carcinogenesis, the roles played by PTKs in mitogenesis and oncogenesis have been most extensively studied. PTKs have also been identified as the translation products of a number of oncogenes. Expression of these tyrosine kinase oncoproteins in cultured cells results in pronounced alterations in cell shape and growth control, and in many cases, renders the transformed cells tumorigenic in syngeneic animals or nude mice. In addition, inoculation of retroviruses containing a tyrosine kinase oncogene into animals often leads to the rapid induction of leukemias or solid tumors. Thus, oncogenic PTKs subvert the regulation of normal cell growth, presumably by chronically transmitting a mitogenic signal into the cell.

Thyroid and growth factors

Although TSH is the major regulator of thyroid growth and function, the role of TSH in thyroid cell growth in vitro is controversial. Recent studies have shown that a number of growth factors, cytokines, vitamins, hormones and other reagents including iodide, lithium or retinoids modulate proliferation of cultured thyroid cells. Insulin-like growth factors (IGFs) stimulate growth of thyroid cells from a variety of species and also enhance expression of differentiated functions stimulated by TSH. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and Transforming growth factor (TGF)-alpha are also mitogens to thyroid cells, but they are inhibitory to differentiated functions. In contrast, TGF-beta is a potent inhibitor to thyroid cell growth and functions. Indirect evidence suggests that these growth factors are involved in growth regulation of normal and neoplastic thyroid cell growth in autocrine/paracrine manner.

Partial characterization of glioma-derived growth factor 2: a novel mitogenic activity from human cell line D-54 MG.

We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111:B4, 1 microgram/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells. This glioma-derived growth factor (GDGF-2) acts like a 'competence' factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serum-free conditioned culture medium. GDGF-2 is resistant to heat (100 degrees C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGF alpha, TGF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 x 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF beta antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha 2-macroglobulin (alpha 2M), which is known to bind TGF beta; however, immunoprecipitation of alpha 2M did not deplete TGF beta or GDGF-2 activity. Further, neither GDGF-2 or TGF beta can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.