蛙皮素对人胃黏膜上皮永生细胞系cyclin D1/CDK4的影响

目的 研究蛙皮素对正常胃黏膜上皮细胞的促生长作用及其与细胞调控因子之间的关系。方法 应用人胃黏膜上皮永生细胞株GES－1,经蛙皮素及受体拮抗剂不同处理后,以MTT为指标研究对细胞增殖的影响;流式细胞仪观察对细胞cyclin D1表达的影响;利用Western印迹和增强化学发光法观察CDK4蛋白表达的变化;利用Sepharose微珠免疫沉淀方法检测对CDK4活性的影响。结果 蛙皮素在浓度为10^-7mol/L时对GES－1细胞有明显的促生长作用,对cyclin D1表达有促进作用;蛙皮素受体拮抗剂均抑制这些作用;同时还发现蛙皮素可使人胃黏膜上皮永生细胞系细胞的CDK4蛋白表达及其激酶活性增加。结论 蛙皮素促进人胃黏膜上皮细胞生长是通过使细胞周期调控因子cyclin D1和CDK4蛋白表达升高,CDK4激酶活性增加,促进细胞周期的进展而实现的。     

Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.     

Genetic changes in the transforming growth factor beta (TGF-beta) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-beta.

We have found several genetic changes in the TGF-beta-type II receptor gene in human gastric cancer cell lines resistant to the growth inhibitory effect of TGF-beta. Southern blot analysis showed deletion of the type II receptor gene in two of eight cell lines and amplification in another two lines. The single cell line we studied that is sensitive to growth inhibition by TGF-beta showed no structural abnormalities of the type II receptor gene. Some of the gastric cancer cells resistant to the growth inhibitory effect of TGF-beta express either truncated or no detectable TGF-beta type II receptor mRNAs, whereas the one that retains responsiveness to the growth inhibitory effect of TGF-beta expresses a full-size type II receptor mRNA. Immunoprecipitation followed by Western blot analysis showed parallel changes in TGF-beta type II receptor expression. Our results suggest that one of the possible mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve genetic changes in the TGF-beta type II receptor gene itself or altered expression of its mRNA.     

Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer

1,25-(OH)(2) vitamin D(3) (1,25-(OH)(2) D), the active metabolite of vitamin D, exerts antiproliferative effects on a variety of tumor cells including prostate. This inhibition requires vitamin D receptors (VDRs) as well as downstream effects on the G1 to S phase checkpoint of the cell cycle. Recent data raise the possibility that androgen plays a role in the antiproliferative effects of 1,25-(OH)(2) D in prostate cancer cells; however, this hypothesis has been difficult to test rigorously as the majority of prostate cancer cell lines (unlike human prostate tumors) lack androgen receptors (ARs). We utilized two different models of androgen-independent prostate cancer that express functional ARs and VDRs to evaluate a possible role of androgen in 1,25-(OH)(2) D mediated growth inhibition. We stably introduced the AR cDNA into the human prostate cancer cell line ALVA 31, which expresses functional VDR but is relatively resistant to growth inhibition by 1,25-(OH)(2) D. Neither ALVA-AR nor the control cells, ALVA-NEO, exhibited substantial growth inhibition by 1,25-(OH)(2) D in the presence or absence of androgen. This observation suggests that the basis for the resistance of ALVA 31 to 1,25-(OH)(2) D-mediated growth inhibition is not the lack of AR. The second model was LNCaP-104R1, an AR-expressing androgen independent prostate cancer cell line derived from androgen dependent LNCaP. 1,25-(OH)(2) D inhibited the growth of LNCaP-104R1 cells in the absence of androgen and this effect was not blocked by the antiandrogen Casodex. As was observed in the parental LNCaP cells, this effect was correlated with G1 phase cell cycle accumulation and upregulation of the cyclin dependent kinase inhibitor (CKI) p27, as well as increased association of p27 with cyclin dependent kinase 2. These findings suggest that the antiproliferative effects of 1,25-(OH)(2) D do not require androgen-activated AR but do involve 1,25-(OH)(2) D induction of CKIs required for G1 cell cycle checkpoint control.     

Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA.

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.     

Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G₁ in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G₁-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G₁ active in the prevention of human bronchial epithelial cell transformation.     

Cellular mechanisms of growth inhibition of human epithelial ovarian cancer cell line by LH-releasing hormone antagonist Cetrorelix

We investigated the direct effects of LH-releasing hormone (LH-RH) antagonist, Cetrorelix, on the growth of HTOA human epithelial ovarian cancer cell line. RT-PCR revealed the expression of mRNA for LH-RH and its receptor in HTOA cells. Cetrorelix, at concentrations between 10(-9) and 10(-5) M, exerted a dose-dependent antiproliferative action on HTOA cells, as measured by 5-bromo-2'-deoxyuridine incorporation into DNA. Flow cytometric analysis indicated that Cetrorelix, at 10(-5) M, arrested cell cycle in HTOA cells, at G1 phase, after 24 h of treatment. Western blot analysis of cell cycle-regulatory proteins demonstrated that treatment with Cetrorelix (10(-5) M) for 24 h did not change the steady-state levels of cyclin D1, cyclin E, and cyclin-dependent kinase (Cdk)4 but decreased the levels of cyclin A and Cdk2. The protein levels of p21 (a Cdk inhibitor) and p53 (a suppressor of tumor cell growth and a positive regulator for p21 expression) were increased by Cetrorelix, but the levels of p27 (a Cdk inhibitor) did not change significantly. Flow cytometric analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick end labeling staining demonstrated that Cetrorelix (10(-5) M) induced apoptosis in HTOA cells. In conclusion, Cetrorelix directly inhibits the proliferation of human epithelial ovarian cancer cells through mechanisms mediated by LH-RH receptor and involving multiple events in cell cycle progression, including G1 phase cell cycle arrest coupled with down-regulation of cyclin A-Cdk2 complex levels, presumably attributable to an up-regulation of p53 and p21 protein levels and apoptosis.     

Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.     

TGF-beta cytokines increase senescence-associated beta-galactosidase activity in human prostate basal cells by supporting differentiation processes, but not cellular senescence

The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.     

Modulation of bcl-2 and p27 in human primitive proliferating hematopoietic progenitors by autocrine TGF-beta1 is a cell cycle-independent effect and influences their hematopoietic potential

Primitive, proliferating hematopoietic progenitors (defined as cytokine low-responding primitive progenitors; CLRPP), isolated from human CD34+ cells, expressed endoglin (CD105) and produced transforming growth factor-beta1 (TGF-beta1). Culture of CLRPP in serum-free conditions with anti-TGF-beta1 monoclonal antibody produced a substantial decrease in bcl-2 protein/RNA levels and a significant reduction of cloning and long-term culture-initiating cell (LTC-IC) activities. GATA-1 and PU.1 RNA levels were significantly up-regulated in anti-TGF-beta1-treated CLRPP, which generated an increased number of cells expressing CD15/CD11b/glycophorin-A. The described effects of TGF-beta1 neutralization were observed in the absence of any relevant effect on cell cycle; number of cell divisions; p53, c-myc, and p21 RNA levels; bcl-xL and bax protein levels; and c-myc/p16/p21/p107/Rb cell cycle-related protein levels. A relevant increase in p27 protein levels was observed in anti-TGF-beta1-treated CLRPP, suggesting a role for p27 in the regulation of the hematopoietic potential. The present study on human progenitors and previously reported data on TGF-beta1 knockout mice suggest that, at the autocrine level, the cell cycle inhibitor TGF-beta1 plays an important role in regulating the survival and differentiation of primitive proliferating hematopoietic progenitors by cell cycle-independent mechanisms.     

Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene.

Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.     

Induction of transforming growth factor-beta 1 (TGF-beta 1), receptor expression and TGF-beta 1 protein production in retinoic acid-treated HL-60 cells: possible TGF-beta 1-mediated autocrine inhibition.

Treatment of HL-60 cells, a human promyelocytic leukemia cell line, with the vitamin A derivative retinoic acid (RA) for 7 days resulted in a dose-dependent decrease in proliferation and increase in granulocytic differentiation. The role of transforming growth factor-beta 1 (TGF-beta 1), a protein with pleiotropic effects on the proliferation and differentiation of various cell types, was examined during RA-induced differentiation of HL-60 cells. Although TGF-beta 1 alone had little effect on proliferation or differentiation of HL-60 cells, addition of TGF-beta 1 to HL-60 cells treated with a suboptimum concentration of RA (1.0 nmol/L) resulted in a marked decrease in proliferation with no effect on granulocytic differentiation. Studies of the mechanism of RA-induced TGF-beta sensitivity showed that although untreated HL-60 cells expressed low levels of TGF-beta 1 binding proteins on the cell surface, the levels were increased in a dose-dependent manner after RA treatment. Maximum induction was achieved after treatment with 10 nmol/L RA and consisted predominantly of the 65-Kd TGF-beta 1 receptor type. Moreover, RA treatment also resulted in a dose-dependent increase in both TGF-beta 1 steady-state mRNA expression and production of active TGF-beta with maximum induction at 10 nmol/LRA. RA treatment of HL-60 cells had no effect on TGF-beta 2 and TGF-beta 3 mRNA expression. These data suggest that the effects of RA may be mediated by a TGF-beta 1-mediated autocrine antiproliferative loop during differentiation of HL-60 cells.     

Gene silencing of ß-catenin by RNAi inhibits cell proliferation in human esophageal cancer cells in vitro and in nude mice

ß-catenin, which is frequently overexpressed in a variety of human cancers including esophageal cancer, mediates cancer cell proliferation and tumor growth. In the present study, we used a human U6 promoter-driven DNA-template approach to induce short hairpin RNA (shRNA)-triggered RNA interference to silence ß-catenin gene expression in human esophageal squamous cell carcinoma cell line Eca-109, and then evaluated its effects on the proliferation and growth of tumor cells in vitro and in nude mice. ß-catenin expression levels decreased markedly in Eca-109 cells transfected with a plasmid expressing shRNA for ß-catenin. Downregulation of ß-catenin was concomitantly accompanied by reduction of cyclin D1, colony formation, and growth inhibition of Eca-109 cells in vitro . The mechanism appears to be the G0/G1 phase arrest but not induction of apoptosis. In vivo , treatment of Eca-109 cells with ß-catenin shRNA greatly impeded tumor growth in nude mice. We conclude that plasmid vector-mediated ß-catenin RNA interference holds great promise as a novel treatment on human esophageal cancer with ß-catenin overexpression.     

Resistance to TGF-beta 1 correlates with aberrant expression of TGF-beta receptor II in human B-cell lymphoma cell lines

Resistance to transforming growth factor (TGF)-beta1-mediated growth suppression in tumor cells is often associated with the functional loss of TGF-beta receptors. Here we describe two B-cell lymphoma cell lines (DB and RL) that differ in their sensitivity to TGF-beta1-mediated growth suppression. The TGF-beta1-resistant cell line DB lacked functional TGF-beta receptor II (T beta RII) in contrast to the TGF-beta-responsive cell line RL, whereas both cell lines had comparable levels of receptor I (T beta RI). Lack of functional T beta RII was correlated with the lack of TGF-beta1-induced nuclear translocation of phospho-Smad3 and phospho-Smad2, the lack of nuclear expression of p21(Cip1/WAF1), and the down-regulation of c-Myc in DB cells. Transfection of wild-type, but not a C-terminal-truncated, form of T beta RII rendered the DB cell line responsive to TGF-beta1-mediated growth suppression. Analysis of the T beta RII gene in DB cells revealed the absence of T beta RII message, which was reversed upon 5'-azacytidine treatment, indicating that the promoter methylation might be the cause of gene silencing. Promoter analysis revealed CpG methylations at -25 and -140 that correlated with the gene silencing. These data suggest that promoter methylation plays an important role in T beta RII gene silencing and subsequent development of a TGF-beta1-resistant phenotype by some B-cell lymphoma cells.