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PubMed1998

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR.

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.

Cloning, Molecular↗

PubMed2004

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display.

OBJECTIVE: To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. METHODS: Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. RESULTS: Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. CONCLUSIONS: Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Adult↗

PubMed2001

Chicken granulosa cells show differential expression of epidermal growth factor (EGF) and luteinizing hormone (LH) receptor messenger RNA and differential responsiveness to EGF and LH dependent upon location of granulosa cells to the germinal disc.

Granulosa cells in the chicken follicle exhibit different phenotypes according to their location relative to the germinal disc (GD). Granulosa cells proximal to the GD (referred to as proximal granulosa cells) are more proliferative, whereas granulosa cells distal to the GD (referred to as distal granulosa cells) are more differentiated. We have shown that epidermal growth factor (EGF) derived from the GD stimulated proliferation of granulosa cells proximal to the GD, whereas extraovarian LH promoted differentiation. We tested the hypothesis that phenotypic differences of granulosa cells are the result of differential responsiveness of granulosa cells to EGF and LH. We found that both granulosa and theca layers of chicken preovulatory follicles expressed mRNA for EGF receptor (EGFr) by polymerase chain reaction (PCR) analysis. However, only the granulosa layer showed differential expression of EGFr and LH receptor (LHr) mRNA. Competitive reverse transcription-PCR revealed that proximal granulosa cells expressed more EGFr mRNA but less LHr mRNA than distal granulosa cells. In addition, proximal granulosa cells proliferated more in response to EGF than their distal counterparts. We further demonstrated that EGF decreased LHr mRNA expression by granulosa cells in a dose-dependent manner, whereas EGF and LH had no effect on EGFr mRNA expression except at one dose of LH (15 ng/ml) that stimulated EGFr mRNA expression. Our findings suggest that EGF derived from the GD influences the phenotypes of granulosa cells. Granulosa cells proximal to the GD exhibit a proliferative phenotype possibly because they are exposed to and are more responsive to GD-derived EGF. Furthermore, GD-derived EGF decreases LHr mRNA expression by proximal granulosa cells and therefore results in less differentiated granulosa cell phenotype. In contrast, granulosa cells distal to the GD are not under the influence of EGF and exhibit a more differentiated phenotype.

Animals↗

PubMed1998

Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistant cell lines: comparison of mRNA differential display reverse transcription-polymerase chain reaction and subtractive suppressive hybridization for the analysis of differential gene expression.

Subtractive suppressive hybridization (SSH) and mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) were compared for their ability to detect the expression of drug-resistance associated genes in a doxorubicin-resistant and -sensitive colon carcinoma cell line (LoVo H67P). The expression pattern of more than 9000 bands obtained by DDRT-PCR were identical in both cell lines by more than 95%. Of the remaining differentially expressed DDRT-PCR products, 21 cDNA fragments were further analyzed after cloning. A total of 210 clones were sequenced resulting in 40 different sequences of which only five were differentially expressed as revealed by Northern blot analysis. SSH, on the other hand, resulted in 30 different sequences of 37 clones analyzed. Thirteen of 30 sequences (43%) could be identified by databank analysis (excluding expressed sequence tags) in contrast to nine of 40 clones (23%) obtained by DDRT-PCR. Of the clones identified by SSH, 60% exhibited a differential expression comparing the doxorubicin-resistant and -sensitive cell line, respectively, as compared to only 13% of the DDRT-PCR derived clones. The application of SSH resulted in the identification of differentially expressed genes in three doxorubin-resistant cell lines (LoVo DxR, ARH D60 and KB-V1) as compared to the sensitive parental cell lines. A significant higher expression of S100P, a protein involved in calcium metabolism, as well as MAGE 3 (melanoma antigen gene) was found in the resistant cell lines using this methodology. The expression of CAPL, a second protein involved in calcium metabolism, was only moderately elevated in the doxorubicin-resistant cells. We found that subtractive suppressive hybridization proved to be a more rapid and reliable method for the detection of differentially expressed mRNAs in our system.

Antigens, Neoplasm↗

PubMed1995

Differential display of messenger ribonucleic acid: a useful technique for analyzing differential gene expression in human brain tumors.

In this study, a differential display method for messenger ribonucleic acid was successfully used to identify genes differentially expressed between normal human brain and malignant glioma tissues. A total of 60 differentially expressed sequences were initially identified, of which 21 were cloned and sequenced. Twenty of the cloned sequences represented novel genes, and one sequence represented a kinesin heavy chain (KHC) gene isoform. The KHC isoform was selected for further characterization. Northern blots of total ribonucleic acid isolated from normal brain and a glioblastoma were probed with our KHC probe and confirmed the differential expression of this gene. Expression analysis of a variety of normal human tissues demonstrated that this KHC isoform is expressed only in brain tissues, with no detectable expression in placenta, spleen, kidney, lung, liver, or skeletal muscle. Our results confirm the rapid and sensitive nature of the differential display technique in identifying differential gene expression. This method offers a means to identify new genes of biological interest in human brain tumors such as oncogenes, tumor suppressor genes, and tumor-specific markers.

Adult↗

PubMed2000

Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR.

Genes that control the specification and differentiation of the functionally specialized areas of the mammalian neocortex are likely expressed across the developing neocortex in graded or restricted patterns. To search for such genes we have performed a PCR-based differential display screen using RNAs from rostral neocortex, which included the primary motor area, and caudal neocortex, which included the primary visual area, of embryonic day 16 rats. We identified 82 differentially expressed gene fragments. Secondary screening by in situ hybridization confirmed that five fragments, representing four genes, are differentially expressed across developing rat neocortex. Two of the genes, chick ovalbumin upstream transcription factor I (COUP-TFI) and close homolog of L1 (CHL1), have been cloned previously, but their differential expression in cortex has not been reported. Sequences from the other two fragments suggest that they represent novel genes. The expression patterns include graded, restricted, and discontinuous expression with abrupt borders that might correlate with those of areas. The differential expression patterns of all four genes are established before the arrival of thalamocortical afferents, suggesting that they are independent of thalamic influence, and could direct or reflect arealization. In addition, COUP-TFI and CHL1 exhibit dynamic expression patterns that undergo substantial changes after thalamocortical afferents invade the cortical plate, suggesting that thalamic axons may influence their later expression. Postnatally, COUP-TFI is most prominently expressed in layer 4, in both rats and mice, and CHL1 is expressed in layer 5. COUP-TFI expression in cortex, and in ventral telencephalon and dorsal thalamus, suggests several possible causes for the loss of layer 4 neurons and the reduced thalamocortical projection reported in COUP-TFI knock-out mice.

Animals↗

PubMed2006

[Identification of the differentially expressed genes between primary breast cancer and paired lymph node metastasis through combining mRNA differential display and gene microarray].

OBJECTIVE: To identify and clone the genes related to breast cancer metastasis through comparing the mRNA expression profiling between primary breast cancer and paired lymph node metastasis. METHODS: First, primary breast cancer cells and paired lymph node metastasis tissues were used to identify their differentially expressed genes by using mRNA differential display, and fragments of differentially expressed genes were obtained by gel cutting and cloning to pGEM-T vector, then sequencing and blasting with the GenBank for their homologous genes. Then, the obtained genes were validated by using reverse dot blot hybridization and gene microarray. Finally, the mRNA expression levels in the 7 breast cancer cell lines with different metastasis behaviors were detected by real-time RT-PCR. RESULTS: The mRNA expression profiling of the metastatic breast cancers in lymph node was similar to that of their primary cancers. 16 differentially expressed gene fragments were obtained from mRNA differential display; four of them were found to be unknown genes, and had been accepted by the GenBank, with the accession numbers BG518428, BG518429, BM005520, and BM005521. Of the other 12 genes, 8 were known genes, and 4 were genes with known sequences but unknown functions. The kinesin-like DNA binding protein (KNSL4) and dihydropyrimidine dehydrogenase (DYPD) were down-regulated in the metastatic tissues in comparison with the paired primary tissues, which was identified by both radioactivity-labeled reverse dot blot hybridization and fluorescence-labeled gene microarray hybridization. KNSL4 mRNA expression had a down-regulation trend with increasing metastatic ability of breast cancer cell lines. CONCLUSIONS: The gene expression profiling of the lymph node metastasis is almost similar to that of their primary breast cancer, indicating that the metastatic cancer cells in lymph node are the subclone with higher metastasis ability of the primary cancer cells, and that some differentially expressed genes between them may be involved in the change of metastasis phenotype. KNSL4 and DYPD are potential metastasis-related genes, and KNSL4 mRNA expression is correlated with metastasis behaviors of the breast cancer cell lines, suggesting that KNSL4 may play an important role in the metastasis process of breast cancer.

Breast Neoplasms↗

PubMed1998

Well-differentiated versus less-differentiated endometrial carcinoma.

The aim of this study was to compare well-differentiated (grade 1) and less-differentiated (grades 2 and 3) endometrial carcinoma, concerning clinical risk factors, presenting symptoms and operative findings. Seventy-one surgically staged endometrial carcinoma cases were reviewed and retrospectively divided according to tumor differentiation into two groups: grade 1 cases (Group I; n=40) and grade 2 and 3 cases (Group II; n=31). No difference was found between the two groups when comparing patient age, gravidity and parity, history of hypertension, diabetes or other malignancies, duration of menopause, number of patients receiving hormonal replacement therapy and histological type of tumor. There was also no difference in presenting symptoms or in duration of uterine bleeding up to the diagnosis of endometrial carcinoma. On the other hand, patients with well-differentiated tumors, as compared to patients with less-differentiated endometrial carcinoma, were significantly more obese (p<0.02), had a smaller uterus (p<0.01) and presented with less advanced stage disease (p<0.0005). The fact that the age of the patients, as well as the duration of uterine bleeding before diagnosis, was similar in both groups may indicate that more advanced cases, as in group II, represent originally a more aggressive tumor rather than progression from a well-differentiated disease. This hypothesis needs further research.

Aged↗

PubMed2001

Differentiation of liver cell adenomas from well-differentiated hepatocellular carcinomas by comparative genomic hybridization.

Liver cell adenomas (LCAs) are rare tumours which may be difficult to differentiate from low-grade hepatocellular carcinomas (HCCs). This study used comparative genomic hybridization (CGH) to look for cytogenetic aberrations which would serve to distinguish between these tumours. For this purpose, ten LCAs and six well-differentiated HCCs were analysed and the results were compared with those reported previously for 15 well-differentiated HCCs. Aberrations were seen in 2/10 LCAs: a gain of chromosome 7p was observed in one and gains of 17q and 20 in a second case. In 6/6 well-differentiated HCCs, up to 13 aberrations were detectable, with a mean of 7.2 aberrations per case in chromosome sites 1q, 4p, 4q, 5p, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 10q, 11p, 13q, 14q, 16p, 16q, 17p, 17q, 20p, 20q, and 21q. Aberrations focused on gains or losses of six chromosome sites, 1q, 4q, 8p, 8q, 16p, and 17p; in all HCC samples, at least two of these sites were affected. None of these aberrations occurred in any of the LCAs analysed. CGH is therefore helpful in distinguishing between LCA and well-differentiated HCC. Detection of one or more of the six most frequent aberrations in HCC supports the diagnosis of carcinoma and makes LCA unlikely.

Adenoma, Liver Cell↗

PubMed2006

Differentiation status of human renal proximal and distal tubular epithelial cells in vitro: Differential expression of characteristic markers.

BACKGROUND: The culture of human renal tubular cells of well-defined nephron origin is an important basis in the research of various physiological and pathophysiological mechanisms in the kidney. In vitro differentiation of cultured cells is affected by cell isolation and culture conditions. Our study describes in vitro differentiation of cultured human renal proximal and distal (thick ascending limb and early distal) tubular epithelial cells. METHODS: Proximal tubular cells (PTC) and early distal tubular cells (DTC) were isolated immunomagnetically and cultured. In vitro differentiation was assessed by Western blot analysis and reverse transcriptase polymerase chain reaction using characteristic markers. Morphologic characterization was shown by fluorescence and scanning electron microscopy. RESULTS: E-cadherin was highly expressed in DTC, whereas expression was low in PTC. In contrast to DTC, in PTC, intercellular adhesion molecule 1 and aquaporin 1 were constitutively expressed at high levels. Both cell types expressed cytokeratin 18 and Na-K-ATPase but not alpha-smooth muscle actin. Morphological analysis showed that PTC develop long microvilli, whereas DTC formed short microvilli in culture, and both express the tight junction protein zona occludens protein 1. CONCLUSIONS: We demonstrate differential expression of E-cadherin, intercellular adhesion molecule 1 and aquaporin 1 in cultured PTC and DTC as described for the renal tubule in vivo. We could show the in vitro differentiation of the cells, and thus, their usefulness as an in vitro model of the human proximal and early distal tubule.

Antigens, Differentiation↗

PubMed2003

Expression of WASP and Scar1/WAVE1 actin-associated proteins is differentially modulated during differentiation of HL-60 cells.

The Wiskott-Aldrich Syndrome (WAS) is a disease associated with mutations in the WAS gene and characterised by developmental defects in haematopoietic cells such as myeloid cells. The Wiskott-Aldrich Syndrome protein (WASP)-family includes Scar1 and WASP, which are key regulators of actin reorganization in motile cells. To understand the roles of Scar1 and WASP in myeloid cells and their cytoskeletal control in haematopoietic tissues, we have explored their expression during differentiation of the promyeloid cell line HL-60. Undifferentiated HL-60 cells expressed Scar1 and WASP, and differentiation to neutrophils, induced by retinoic acid or non-retinoid agent treatments, led to a decrease in the level of expression of Scar1, whereas WASP expression was unaffected. Differentiation to monocytes/macrophages, induced by phorbol ester treatment, resulted in a decreased expression of both proteins in the adherent mature cells. Vitamin D(3) treatment or cytochalasin D in combination with PMA treatment did not affect WASP expression suggesting that adhesion and cytoskeletal integrity were both essential to regulate WASP expression. Scar1 expression was regulated by differentiation, adhesion, and cytoskeletal integrity. Recently, WASP was found to colocalize with actin in the podosomes. In contrast, we show here that Scar1 did not localize with the podosomes in mature monocytes/macrophages. These observations show for the first time that modulation of Scar1 and WASP expression is a component of the differentiation program of myeloid precursors and indicate that WASP and Scar1 have different roles in mature myeloid cells.

Actin Cytoskeleton↗

PubMed2003

Vesicle-associated protein-A is differentially expressed during intestinal smooth muscle cell differentiation.

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes. This analysis identified over 40 unique transcripts that appeared to be differentially expressed in distinct SMC phenotypes. One such transcript that appeared to be preferentially expressed in immature smooth muscle myocytes was identified as vesicle-associated membrane protein, associated protein A (VAP-A). Northern blot analysis confirmed that VAP-A was expressed threefold higher in immature smooth muscle myocytes when compared with both smooth muscle myoblasts and mature smooth muscle myocytes. VAP-A mRNA was differentially expressed during normal rat development and showed peak levels of expression in the intestine during late embryogenesis and early neonatal development. These observations provide the first evidence that VAP-A-mediated membrane trafficking may play an important role in modulating ISMC differentiation.

Amino Acid Sequence↗

PubMed1986

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle.

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1985

Induction of cell differentiation in Burkitt lymphoma lines. BLA: a glycolipid marker of B-cell differentiation.

We have previously described an MAb referred to as 38.13 which reacts with a glycolipid membrane antigen (named BLA) on Burkitt lymphoma (BL) lines. The BLA antigen and other B-cell differentiation markers have been studied on BL lines treated with sodium butyrate, agents from the phorbol-diester series (with or without differentiating properties) and teleocidin. With all differentiation inducers tested, expression of BLA as well as of surface IgM decreased on the induced cells whereas that of BI increased and that of HLA DR remained stable. No BL cells studied reacted with anti-IgD, with B2 or with LBI MAbs, either before or after induction. An EBV-negative line (BJAB) was compared to its Epstein-Barr virus (EBV)-converted subline (BJAB/B95). Both EBV-positive and EBV-negative lines gave comparable results. These data demonstrate that BL cells could be moved along their differentiation pathway by chemical inducers and suggest that BLA represents a new glycolipid marker of early B-cell differentiation.

Antibodies, Monoclonal↗

PubMed2006

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES.

Chemokines MCP-1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP-1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1alpha is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1alpha was able to overcome the potent inhibition of GM-CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6- and 49-fold, P = 4.0 x 10(-7) and 4.0 x 10(-8), respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP-1, RANTES and MIP1alpha. Unexpectedly, treatment with MCP-1 in the absence of RANKL resulted in 458-fold induction of CCR4 (P = 1.0 x 10(-10)), while RANTES treatment resulted in twofold repression (P = 1.0 x 10(-4)). Since CCR2b and CCR4 are MCP-1 receptors, these data support the existence of an MCP-1 autocrine loop in human osteoclasts differentiated using RANKL.

Acid Phosphatase↗

PubMed1989

Differential effect of dexamethasone on the regulation of phospholipase A2 and prostanoid synthesis in undifferentiated and phorbolester-differentiated U937 cells.

The human undifferentiated histiocytic cell-line U937 can be induced to differentiate by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells. Dexamethasone reduced the prostaglandin production in TPA-differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA-differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA-differentiated U937 cells. The data suggest that responsiveness to glucocorticoid-dependent regulation of prostanoid synthesis is acquired by cells of the monocyte-macrophage lineage late in differentiation.

Acyltransferases↗

PubMed2002

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated, growth-arrested U937 cells, overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.

Cell Cycle↗

PubMed1986

N-myc and c-src genes are differentially regulated in PCC7 embryonal carcinoma cells undergoing neuronal differentiation.

We examined the expression of N-myc, c-myc, and c-src in four embryonic carcinoma (EC) cell lines during different states of cell growth and following induction of in vitro differentiation. N-myc mRNA was detected in undifferentiated cells of four EC cell lines (PCC7, PCC3, PCC4, F9) neither of which showed N-myc gene amplification. No N-myc transcripts could be detected in mRNA prepared from a murine neuroblastoma cell line and from a murine fibroblast line. The level of N-myc mRNA decreased by 85% when PCC7 EC cells were induced by retinoic acid and cAMP treatment to form nerve-like cells. Six days after induction, the PCC7 cells changed into aggregates of neurofilament positive cells with massive neurite outgrowths. At this stage DNA replication had been reduced by more than 95%. The decreased N-myc expression in induced PCC7 cells was parallelled by 300-500% increase in c-src expression. Slowing of cell multiplication by serum starvation, on the other hand, did not affect the level of N-myc or c-src mRNA levels in PCC7 cells. C-myc was expressed in all EC lines except PCC7, which surprisingly did not express c-myc even at an exponential rate of proliferation. Chemical induction of F9 EC cells to form visceral endoderm or parietal endoderm resulted in markedly reduced (85%) levels of N-myc transcripts. A similar decline in c-myc expression was found in differentiated F9 cells. No c-src transcripts were detected in proliferating or differentiated F9 cells. These results suggest that N-myc may be expressed not only in neural development, but also in very early, undetermined embryonic cells. The activation of c-src expression when PCC7 EC cells differentiate into nerve-like cells shows that the pattern of proto-oncogene expression may change during a differentiation process, some proto-oncogenes increasing, others decreasing their representation in the mRNA pool.

Blood↗