Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation.

The activities of the three known DNA polymerases-alpha, beta-, and -gamma were determined in rat brain neurons, cardiac muscle and spleen, and were correlated with the rate of cell proliferation during perinatal development. In neurons and cardiac muscle, which stop dividing before birth, DNA polymerase-alpha activity drops sharply from a high level with the approach of term and disappears at approximately two weeks postnatal age. In contrast, alpha-polymerase activity is almost absent in spleen during late gestation, when the rate of cell division is low, and increases abruptly after birth with the sudden onset of cell proliferation. These data give further evidence for an involvement of DNA polymerase-alpha in DNA replication. DNA polymerase-beta and -gamma activities show essentially no correlation with the rate of cell division. Thus, these enzymes are probably responsible for repair type processes rather than for DNA replecation.     

Regulation of cell differentiation by the DNA damage response

When faced with DNA double-strand breaks (DSBs), vertebrate cells activate DNA damage response (DDR) programs that preserve genome integrity and suppress malignant transformation. Three established outcomes of the DDR include transient cell cycle arrest coupled with DNA repair, apoptosis, or senescence. However, recent studies in normal and cancer precursor or stem cells suggest that a fourth potential outcome, cell differentiation, is under the influence of DDR programs. Here we review and discuss the emerging evidence that supports the linkage of signaling from DSBs to the regulation of differentiation, including some of the molecular mechanisms driving this under-appreciated DDR outcome. We also consider the physiologic and pathologic consequences of defects in DDR signaling on cell differentiation and malignant transformation.     

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.     

YTHDF1 promotes breast cancer cell growth,DNA damage repair and chemoresistance

Chemoresistance represents a major obstacle to the treatment of human cancers. Increased DNA repair capacity is one of the important mechanisms underlying chemoresistance. In silico analysis indicated that YTHDF1, an m6A binding protein, is a putative tumor promoter in breast cancer. Loss of function studies further showed that YTHDF1 promotes breast cancer cell growth in vitro and in vivo. YTHDF1 facilitates S-phase entry, DNA replication and DNA damage repair, and accordingly YTHDF1 knockdown sensitizes breast cancer cells to Adriamycin and Cisplatin as well as Olaparib, a PARP inhibitor. E2F8 is a target molecule by YTHDF1 which modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. These data demonstrate that YTHDF1 has a tumor-promoting role in breast cancer, and is a novel target to overcome chemoresistance.Subject terms: Breast cancer, Breast cancer     

Mechanism of unbalanced growth-induced cell damage. II. A probable relationship between unbalanced growth, DNA breakage and cell death

This study examines the relationship between unbalanced growth, DNase II activity, DNA breakage and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR) or HU with excess of four deoxyribonucleosides (dNR). It has been found that in the cells arrested by HU or dThR, but still appearing viable with the trypan blue exclusion test, Protein/DNA imbalance and abnormal cell volume are correlated with enhancement of DNase II activity in the cells and in the medium and with moderate increase in parental DNA breakage. The incidence of DNA breaks was markedly potentiated in the presence of non-toxic concentration of caffeine (CAF), used to inhibit DNA repair. In HU+dNR arrested cells, in which unbalanced growth was abolished, enhancement of DNase II activity and of DNA breakage in the presence or absence of CAF was substantially prevented. Comparison of posttreatment cell survival in the presence or absence of CAF confirmed the differential effect of CAF: while in HU or dThR arrested cells the presence of CAF induced marked cell killing, in HU+dNR arrested cells the influence of CAF was negligible. Only a slight effect of CAF was observed in cells in which dThR-induced arrest and unbalanced growth were reversed by deoxycytidine (dCR) addition. It is suggested that the involvement of DNA nucleases in the unbalanced growth-induced overproduction of numerous hydrolytic enzymes, with their progressive leakage through the cell membranes, can lead to progressive DNA digestion. DNA breaks produced in this way are normally, at least partly, repaired. Concomitant exposure of such cells to DNA repair inhibitor can markedly enhance the level of breaks, leading to potentiation of unbalanced growth-induced cell killing.     

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation.

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E2, F2alpha and I2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP1, EP3, and EP4, and examined the expression of mRNAs encoding the three isoforms (alpha, beta, and gamma) of the EP3 receptor. Altogether the results show that the expression of IP, FP, EP1, and EP4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the alpha, beta, and gamma isoforms of the EP3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI2, PGF2alpha, and PGE2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP3 receptor isoforms is in agreement with the well known effect of PGE2 on adenylate cyclase and lipolysis in mature adipocytes.     

Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial-derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization; and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited; and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.     

Influence of fibroblast growth factor, insulin-like growth factor I, and transforming growth factor beta on satellite cell type I collagen expression and localization during differentiation

Expression, and temporal and spatial distribution of type I collagen were investigated in chicken satellite cell cultures during differentiation. There was no difference in the relative amounts of type I collagen after treatment with basic fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-I), or transforming growth factor beta 1 (TGF-beta 1). However, myotube morphology was influenced by the presence of the growth factors. The temporal and spatial distribution of type I collagen was also modified. Control cultures maintained a predominant distribution of type I collagen surrounding the cellular area until approximately 48 h after the initiation of fusion whereas cultures with FGF or IGF-I maintained a cellular localization of type I collagen throughout the fusion process. TGF-beta 1 resulted in the early formation of an extracellular network of type I collagen preceding control cultures by approximately 24 h. These results suggest that type I collagen expression but not localization is independent of satellite cell proliferation and differentiation.     

Constitutive expression of parathyroid hormone-related peptide PTHnP stimulates growth and inhibits differentiation of CFK2 chondrocytes

We have examined the effects of constitutive expression of PTHrP on the growth and differentiation of populations of cells derived from a clonal chondrocytic cell line, CFK2. Cells were stably transfected with cDNA encoding either full-length, secretory PTHrP (CFK2P) or nonsecretory PTHrP (CFK2P-SS). In cultures of cells plated at low density, secretory PTHrP acted as a potent mitogen compared with nonsecretory PTHrP or exogenous PTHrP-(1-34), both of which stimulated only a minor increase in proliferation. In populations of control cells maintained postconfluent for several weeks, there was a dramatic increase in expression of mRNA for type II collagen, aggrecan, and link protein. Addition of exogenous PTHrP-(1-34) at a concentration of 10⁻⁸ M to these cultures was ineffective in inhibiting this time-dependent increase in expression of matrix proteins. In contrast, populations of cells producing either secretory or nonsecretory forms of PTHrP, maintained over the same time period, demonstrated an almost complete inhibition of mRNA expression for matrix proteins. These observations demonstrate that PTHrP acts as a bifunctional modulator of chondrogenesis and that some of its biological activity is exerted via a mechanism distinct from the recognised signal transduction pathways linked to the PTH/PTHrP receptor. © 1996 Wiley-Liss, Inc.     

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1alpha, IL-1beta, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.