Wild-type p53 is not sufficient for serum starvation-induced apoptosis in cancer cells but accelerates apoptosis in sensitive cells

According to the conflicting growth signal model, cells that are driven to proliferate by certain oncogenes undergo apoptosis but not growth arrest upon withdrawal of growth factors. However, we found that the majority of human cancer cell lines continued to proliferate and did not undergo apoptosis following serum withdrawal. As an exeption, wild-type (wt) p53-expressing HCT116 human colon cancer cells underwent apoptosis within 24-36 h of serum deprivation. p53 degradation in human papilloma virus EG-expressing HCT116 cells led to enhanced survival that was not due to growth arrest. These results are consistent with a role for p53 in starvation-induced death in HCT116 cells. However, other cell lines did not undergo apoptosis despite their expression of wt p53. Thus, H460 cells (wt p53) were resistant to starvation-induced death but introduction of the adenovirus EIA oncoprotein induced p53 and also increased sensitivity to serum withdrawal. p53 was not stabilized by E1A and resistance to starvation-induced cell death was observed in E6-expressing H460 cells. These results suggest that although p53 contributes to starvation-induced apoptosis in sensitive (HCT116 and E1A-expressing H460) cancer cell lines, most cancer cells survived despite the presence of wt p53. We conclude that naturally selected human cancer cell lines suppress apoptosis due to conflicting growth signals.     

Loss of XIAP sensitizes rosiglitazone-induced growth inhibition of colon cancer in vivo

Ligands for peroxisome proliferator-activated receptor gamma (PPAR gamma) possess anticancer properties. However, the efficacy of PPAR gamma ligands varies in different cancers. In colon cancer, the role of PPAR gamma and its ligands is controversial. We recently showed that downregulation of X-linked inhibitor of apoptosis protein (XIAP) could sensitize colon cancer cells to troglitazone, and 15-deoxy-D12,14-prostaglandin J2 (15-PGJ2) induced cell killing. In our study, we aimed to examine whether rosiglitazone, another more clinically relevant PPAR gamma ligand, has any synergistic anticancer effect with XIAP downregulation in colon cancer. Human colon cancer cell lines HCT116-XIAP(+/+) cells and HCT116-XIAP(-/-) cells were treated with various concentrations of rosiglitazone. The effects of rosiglitazone on cell proliferation, apoptosis and growth of xenograft colon cancers were studied. Rosiglitazone barely suppressed the growth and only very weakly induced apoptosis in HCT116 cells in vitro. Loss of XIAP did not sensitize HCT116 cells to rosiglitazone-induced growth inhibition or apoptosis. In vivo studies revealed that rosiglitazone strongly suppressed the growth of xenograft colon cancer, especially tumors derived from HCT116-XIAP(-/-) cells. The rosiglitazone-treated tumor had reduced expression of ki-67 and lowered mitotic rate. Downregulation of XIAP was associated with an impaired activation of PPAR gamma by its ligand. Rosiglitazone induced marked upregulation of PTEN in HCT116-XIAP(-/-) cells, as well as in xenograft tumors derived from HCT116-XIAP(-/-) cells. We concluded that rosiglitazone significantly suppresses the growth of xenograft colon cancer, and downregulation of XIAP sensitizes the xenograft tumors to rosiglitazone-induced tumor suppression in vivo via upregulation of PTEN.     

NDUFA4过表达对人结直肠癌细胞体外生长的影响

 目的 探讨过表达NDUFA4对人结直肠癌细胞体外生长的影响及其机制。方法 将p-NDUFA4重组质粒（p-NDUFA4组）和p-Cont对照质粒（p-Cont组）体外分别转染人结直肠癌HCT116细胞；采用Real-time PCR和Western blot检测细胞中NDUFA的表达；CCK-8法和克隆形成实验分别检测HCT116细胞增殖活性和克隆形成能力；实时荧光定量PCR法检测HCT116细胞中CDK2、CDK3、CDK4、CDK6的mRNA水平；Western blot法检测细胞中蛋白总AKT、ERK1/2以及磷酸化AKT（p-AKT）、ERK1/2（p-ERK1/2）的表达水平。结果 与p-Cont对照组相比，p-NDUFA4组中NDUFA4的表达水平显著升高（均P<0.01）；细胞增殖和克隆形成能力明显增强（均P<0.05）；CDK2、CDK3等的mRNA水平显著上调（均P<0.05）；p-NDUFA4组中p-AKT和p-ERK1/2蛋白的表达水平明显上调（均P<0.05）。结论 过表达NDUFA4能明显促进人结直肠癌细胞的体外生长，其机制可能与AKT和ERK信号通路的变化相关。     

Selenomethionine induces p53 mediated cell cycle arrest and apoptosis in human colon cancer cells

While there is an increasing interest in selenium chemoprevention against human colon polyp recurrence and other cancers, the mechanism(s) by which these agents inhibit carcinogenesis are uncertain. Some of the proposed mechanisms include the inhibition of cytosine methyltransferases, carcinogen bioactivation, and inhibition of cyclooxygenase (COX). More recently, it has been suggested that selenium may exert growth inhibitory effects by activating p53. However, the molecular mechanisms of action of selenomethionine, an organoselenium compound present in selenized yeast and currently being investigated in human clinical trials for colon polyp prevention, are unclear. In the present study we tested the hypothesis that selenomethionine might affect colon cancer cell growth by p53 mediated apoptosis and/or cell cycle regulation. Four human colon cancer cell lines including HCT116 and RKO (wild type p53), HCT116-p53KO (isogenic control of HCT116 cells with p53 knocked out) and Caco-2 (mutant p53) were treated with 0-100 microM of selenomethionine for 24, 48 and 72 h. Cell viability rates were determined by the MTT assay. Cell cycle analysis was performed by flow cytometry and apoptosis measured by Annexin V-Cy5 staining. Expression of p53 protein was determined by Western blotting and immunofluorescence assays. All cell lines showed concentration and time dependent growth inhibition with selenomethionine, although HCT116 and RKO cells were the most sensitive to such treatments. Interestingly, although HCT116 and HCT116-p53KO are isogenic cell lines, selenomethionine caused a G2/M cell cycle arrest in HCT116 and RKO cells, but not in HCT116-p53KO cells. Similarly, both HCT116 and RKO demonstrated a significant increase in apoptosis (100-170%; p < 0.01) with 50-100 microM selenomethionine. Cell cycle arrest and apoptosis observed in HCT116 and RKO cell lines were accompanied by a marked increase in p53 protein expression following selenium treatment. These results clearly suggest that selenomethionine exerts p53 dependent growth inhibitory effects in colon cancer cells by inducing G2/M cell cycle arrest as well as apoptosis.     

p21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.     

lncRNA AK126393对结肠癌细胞的抑制作用及机制研究

目的:探索长链非编码 RNA（long non-coding RNA,lncRNA）AK126393调控人结肠癌细胞凋亡的作用及其机制。方法:采用荧光实时定量聚合酶链反应检测人正常结肠上皮细胞和不同结肠癌细胞系中lncRNA AK126393的表达水平,采用pcDNA3.1-AK126393转染HCT116细胞增加AK126393的表达,采用MTT法检测细胞的增殖活力,采用流式细胞术检测细胞凋亡水平,采用Western blotting检测细胞中目标蛋白的表达水平。结果:与人正常结肠上皮细胞系永生化结肠上皮细胞系NCM460相比,人结肠癌细胞系中AK126393的表达均出现显著下降,其中HCT116细胞中AK126393表达最低;pcDNA3.1-AK126393转染HT116细胞后AK126393的相对表达水平出现显著升高;AK126393 的过表达抑制HCT116细胞的增殖能力,促进其细胞凋亡水平,并且引起细胞自噬的发生;同时AK126393 的过表达显著抑制HCT116细胞内AKT/ERK1/2信号通路的磷酸化水平。结论:LncRNA AK126393具有抑制结肠癌细胞增殖、促进凋亡以及引起细胞自噬的作用,其作用可能是通过抑制AKT/ERK1/2信号通路的激活来实现。     

Cytotoxicity and mutagenicity of frameshift-inducing agent ICR191 in mismatch repair-deficient colon cancer cells

BACKGROUND: Deficiency of DNA mismatch repair is a common feature of cancers exhibiting instability of microsatellite DNA sequences. Cancers with microsatellite instability are recognizable by their high rate of spontaneous frameshift mutations within microsatellite sequences, their resistance to killing by cytotoxic agents, and their localization to specific tissues, e.g., the proximal colon and stomach. We hypothesized that the mismatch repair deficiency of these cancers would make them vulnerable to environmental or chemical frameshift-inducing agents. This study was undertaken to test whether exogenous frameshift-inducing agents selectively induce mutations in mismatch repair-deficient cells of mutagen-exposed tissues like the colon and whether cytotoxic doses of these agents would preferentially kill those cells. METHODS: Cytotoxicity of the acridine mutagen 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxy-acridine (ICR191), a DNA frameshift inducer, was determined in the mismatch repair-deficient human colon carcinoma cell line HCT116 versus the repair-reconstituted derivative HCT116+C3. Vulnerability to the mutagenic effects of ICR191 was determined by transfection of HCT116 or HCT116+C3 cells with a frameshift reporter vector, followed by treatment of the cells with ICR191. Alternatively, the reporter vector was reacted ex vivo with ICR191, and the derivatized vector was then transfected into HCT116 or HCT116+C3 cells. RESULTS: ICR191 proved to be fivefold to 10-fold more potent in inducing mutations in mismatch repair-deficient HCT116 cells than in mismatch repair-proficient HCT116+C3 cells. Moreover, at cytotoxic doses of ICR191, repair-deficient HCT116 cells proved to be fivefold more vulnerable to killing than did HCT116+C3 cells. CONCLUSIONS: Frameshift-inducing mutagens can selectively induce mutations in mismatch repair-deficient cells versus mismatch repair-proficient cells. Environmental exposures may, therefore, favor development of cancers with microsatellite instability in tissues like the gut. Frameshift-inducing agents can, however, also preferentially kill mismatch repair-deficient cancer cells and, thus, may be promising as model therapeutic compounds.     

Induction of a less aggressive phenotype in human colon carcinoma HCT116 cells by chronic exposure to HDAC inhibitor SAHA

Histone deacetylase inhibitors (HDACis) are anticancer molecules that epigenetically modulate cell functions. Chronic exposure of HCT116 colon cancer cells to SAHA has been investigated for a better understanding of resistance mechanisms but, surprisingly, a less aggressive tumor phenotype both in vitro and in vivo was obtained after exposure to increasing concentrations of SAHA. Indeed, HCT116/SAHA cells when injected into nude mice showed a reduced engraftment and growth with respect to HCT116 cells. This difference was not observed inoculating the cells into NOD/SCID mice that, differently from nude mice, lack NK activity, thus suggesting the involvement of the native immune response in impairment of HCT116/SAHA cell growth. In agreement with this result, a growing induction of NKG2D ligand expression, MICA and MICB, that are molecular mediators of NK cell killing, was confirmed in HCT116/SAHA chronically exposed to SAHA. A reduced clonogenic efficiency was also observed in HCT116/SAHA with respect to HCT116 cells. Interestingly, even after chronic exposure to SAHA, HCT116/SAHA cells developed only a moderate resistance to SAHA both in vitro and in vivo and they acquired a collateral sensitivity to anthracyclines. These results are of note and probably rely on the fact that, having simultaneously many different targets, HDACis would require many different mutations to display high resistance index. Moreover, to understand the molecular basis of HCT116/SAHA cell phenotype a gene expression profile of cancer genes was evaluated in HCT116 incubated with SAHA for 24 h and in HCT116/SAHA cells to identify selectively regulated genes.     

miRNA-34 a对人结肠癌细胞 HCT116增殖及侵袭转移的影响

目的：研究微小RNA-34a（microRNA-34a,miR-34a）在人结肠癌细胞株HCT116中的过表达对细胞增殖和侵袭的影响及其机制。方法设计合成并构建携带绿色荧光蛋白（ GFP）的miR-34a真核表达载体,脂质体法稳定转染HCT116细胞。通过Realtime PCR验证转染后miR-34a的表达变化。 MTT法检测转染前后HCT116细胞增殖能力变化, Transwell法检测转染前后HCT116细胞侵袭能力改变,western blot检测目的蛋白的表达。结果 miR-34a转染HCT116细胞后其表达量增加到（7．32±1．34）倍,而HCT116细胞增殖能力也受到显著抑制,下降到（0．49±0．10）;Bcl-2蛋白受到miR-34a表达的抑制,而BAX的表达则受到miR-34a表达的增强;HCT116细胞侵袭能力在miR-34a过表达后显著增强,相应的MMP-2和MMP-9表达也出现显著增强。阴性对照组与空白对照组比较无显著性差异。结论 miR-34a的过表达能够抑制人结肠癌细胞HCT116的增殖及侵袭转移,与调控Bcl-2／BAX和MMP-2和-9蛋白的表达密切相关。     

The in vitro and in vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific inhibitor of phosphatidylinositol 3'-kinase, in human colon cancer cells.

PURPOSE: Phosphatidylinositol 3'-kinase (PI3K) and Akt/protein kinase B(PKB) allow for escape from apoptosis in various human cancer cells. We postulated that 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a PI3K inhibitor, should inactivate Akt/PKB, consequently inhibiting cell proliferation and inducing apoptosis in vitro and in vivo. EXPERIMENTAL DESIGN: Human colon cancer cell lines (DLD-1, LoVo, HCT15, and Colo205) and their mouse xenografts (DLD-1 and LoVo) were used in this study. The expression of phosphorylated Akt (Ser(473)) and apoptosis in cancer cells were determined by immunoblotting and immunohistochemistry. To evaluate the activity of caspase-3 in culturing cells, the caspase colorimetric assay was also performed. RESULTS: LY294002 demonstrated a remarkable growth-inhibitory and apoptosis-inducing effect in these colon cancer cell lines, with decreased expression of phosphorylated Akt (Ser(473)). However, there was a great discrepancy between the sensitivity for LY294002 and the level of expression of phosphorylated Akt. Although the LoVo and Colo205 cells exhibited high sensitivity to LY294002 with increased apoptosis, the DLD-1 and HCT15 cells did not show rapid induction of apoptosis. The caspase-3 activity was significantly high in the LoVo cells but not in the DLD-1 cells. In the experiments using mouse xenografts, we found that LY294002 administration in vivo also resulted in suppression of tumor growth and induction of apoptosis, especially in the LoVo tumors, and therefore showed remarkable effectiveness in the mouse peritonitis carcinomatosa model. CONCLUSIONS: PI3K-Akt/PKB plays an important role in colon cancer development and progression by helping to promote cell growth and allowing cells to escape apoptosis. These results propose the usefulness of LY294002 as an antitumoral agent for patients with colorectal cancer.     

Gelam Honey and Ginger Potentiate the Anti Cancer Effect of 5-FU against HCT 116 Colorectal Cancer Cells

The development of chemopreventive approaches using a concoction of phytochemicals is potentially viablefor combating many types of cancer including colon carcinogenesis. This study evaluated the anti-proliferativeeffects of ginger and Gelam honey and its efficacy in enhancing the anti-cancer effects of 5-FU (5-fluorouracil)against a colorectal cancer cell line, HCT 116. Cell viability was measured via MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium) assay showing ginger inhibiting the growthof HCT 116 cells more potently (IC50 of 3mg/mL) in comparison to Gelam honey (IC50 of 75mg/mL). Combinedtreatment of the two compounds (3mg/mL ginger+75mg/mL Gelam honey) synergistically lowered the IC50 ofGelam honey to 22mg/mL. Combination with 35 mg/mL Gelam honey markedly enhanced 5-FU inhibitingeffects on the growth of HCT 116 cells. Subsequent analysis on the induction of cellular apoptosis suggestedthat individual treatment of ginger and Gelam honey produced higher apoptosis than 5-FU alone. In addition,treatment with the combination of two natural compounds increased the apoptotic rate of HCT 116 cells dosedependentlywhile treatment of either ginger or Gelam honey combined with 5-FU only showed modest changes.Combination index analysis showed the combination effect of both natural compounds to be synergistic in theirinhibitory action against HCT 116 colon cancer cells (CI 0.96 < 1). In conclusion, combined treatment of Gelamhoney and ginger extract could potentially enhance the chemotherapeutic effect of 5-FU against colorectal cancer.     

Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines

At present, there is a lack of availability of differentiation markers for colon carcinoma. This may, in part, be a consequence of the diversified function of the normal human colon. This study addresses the possibility that the expression of urokinase and its receptor is inversely related to differentiation in colon carcinoma. Six colon carcinoma cell lines including three well-differentiated (CBS, GEO, FET) and three poorly differentiated ones (HCT116, HCT116b, RKO) were screened for urokinase receptor display and secretion of the plasminogen activator. A radioreceptor assay was used to determine receptor levels. Binding of radioactive urokinase to colon cells was saturable, specific, and time dependent. Cell-bound 125I-labeled protease was unaffected by the presence of epidermal growth factor, low-molecular-weight urokinase, plasminogen, or transferrin. Time course studies revealed that maximum amounts of radioactive tracer were bound in a 30-min period with no change occurring over the course of a 90-min incubation. Scatchard analysis of ligand binding indicated that the well-and poorly differentiated cells could be separated on the basis of receptor display; the aggressive RKO, HCT116, and HCT116b expressed in excess of 10(5) sites per cell, while the more indolent CBS, GEO, and FET possessed less than 1.5 X 10(4) receptors per cell. The colon carcinoma cells were also analyzed for urokinase in the conditioned medium. Low levels of the plasminogen activator (0.8 to 1.3 ng/ml/10(6) cells/72 h) were associated with the more "mature" cells. This was in contrast to the elevated levels of the protease (3.9 to 11.4 ng/ml/10(6) cells/72 h) present in the medium derived from the more aggressive cells (HCT 116, HCT116b, RKO). Thus, secreted urokinase and/or the expression of cellular receptor for the plasminogen activator may provide useful measurements of the degree of undifferentiation of in vitro colon carcinoma.     

Ku70乙酰化修饰介导曲古霉素A（TSA）促结肠癌细胞凋亡的作用机制研究

目的:探讨Ku70乙酰化修饰介导曲古霉素A（TSA）促结肠癌细胞凋亡的作用和机制。方法:选取结肠癌HCT116细胞和SW620细胞，体外培养并采用浓度梯度TSA处理。MTT法观察TSA对细胞的IC50和活力的影响；Western blot和免疫荧光染色观察TSA对结肠癌HCT116细胞和SW620细胞中Ku70、acetyl-Ku70蛋白水平及胞内分布的影响；流式细胞术检测TSA对结肠癌HCT116细胞和SW620细胞凋亡的影响；Western blot检测TSA对结肠癌HCT116细胞和SW620细胞中凋亡相关蛋白Caspase-3、Bax和Bcl-2表达的影响。结果:MTT结果显示TSA可浓度依赖性抑制结肠癌HCT116细胞和SW620细胞活力且其IC50值分别为1.023 μmol/L和1.076 μmol/L（P<0.05）；Western blot和免疫荧光染色结果显示TSA可显著上调结肠癌HCT116细胞和SW620细胞中acetyl-Ku70的蛋白水平并促进其核转入（P<0.05）；流式细胞术结果表明TSA可促进结肠癌HCT116细胞和SW620细胞凋亡（P<0.05）；此外，Western blot结果显示TSA可明显上调结肠癌HCT116细胞和SW620细胞中凋亡蛋白Caspase-3和Bax的表达，下调抗凋亡蛋白Bcl-2的表达（P<0.05）。结论:结肠癌HCT116细胞和SW620细胞中，TSA可能通过增强Ku70乙酰化并促进其核转入，发挥促凋亡作用，为以Ku70翻译后修饰调控为靶点的肿瘤治疗提供新策略和理论依据。     

In vitro efficacy of a novel chemoradiopotentiator--taxoltere metro

PURPOSE: To evaluate the in vitro cytotoxic and radiopotentiating effects of a novel paclitaxel analog (taxoltere metro) on Chinese hamster ovary (CHO) cells and human colon cancer cells. METHODS AND MATERIALS: Three cell lines (CHO cells, HCT116 human colon carcinoma cells [paclitaxel-sensitive], and VM46 cells [paclitaxel-resistant subline of HCT116]) were employed in this study. Cell survival was determined using the standard colony-forming assay. The ID50 value (drug concentrations required to reduce colony formation to 50% of the control value) was determined as a cytotoxic index from each cell survival curve. The sensitizer enhancement ratio (SER) as a radiopotentiating endpoint was determined as the ratio of the D0 values (with or without drugs) under hypoxic or air conditions. RESULTS: Taxoltere metro was 5-15 times more effective in killing CHO cells than paclitaxel under both hypoxic and euoxic treatment conditions. Cytocidal effects of taxoltere metro on HCT116 cells and VM46 cells were 28 and 70 times higher than those of paclitaxel (p<0.001), respectively. Taxoltere metro also produced significant radiopotentiating effects on euoxic CHO and HCT116 cells, but not on hypoxic cells. The SER value of taxoltere metro for CHO cells was about 2.3 at a dose of 100 nM. With HCT116 cells, taxoltere metro yielded an SER of 1.2 at the low dose of 10 nM. In contrast, the parent compound paclitaxel yielded little or no radiosensitization with either CHO or HCT116 cells. CONCLUSION: The results demonstrate that taxoltere metro is significantly more potent than paclitaxel in chemoradiopotentiating CHO cells and HCT116 human colon carcinoma cells. The data strongly suggest that taxoltere metro could be a promising chemoradiopotentiating agent for treatment of cancer.     

Biglycan及FAK信号通路对结肠癌细胞增殖能力的影响及分子机制

目的 研究Biglycan及FAK信号通路对结肠癌细胞增殖的调控作用及其分子机制。方法 构建Biglycan过表达的人结肠癌HCT116细胞并采用FAK抑制剂处理。分为5组:正常对照组、空载体对照组、空载体抑制剂处理组、Biglycan过表达组和Biglycan过表达抑制剂处理组,处理时间为24h。通过Western blot及MTT检测的方法,观察各组HCT116细胞的增殖能力以及FAK、p-FAK、PCNA、p53的表达情况。结果 过表达Biglycan能够显著促进HCT116细胞的增殖以及FAK的磷酸化(P＜0.01),并导致PCNA表达水平的显著升高和p53表达水平的显著抑制(P＜0.01);而FAK抑制剂PF-562271作用能够明显抑制细胞的增殖能力,Biglycan对p-FAK、PCNA、p53蛋白表达水平的调控被抑制(P＜0.01)。结论 Biglycan通过促进FAK信号通路的活化调控结肠癌细胞的增殖。     

人PD-L1真核表达载体的构建及结肠癌细胞稳定表达单克隆株的筛选和鉴定

目的:构建人程序性死亡配体1（PD-L1）重组质粒载体,筛选稳定高表达人PD-L1的结肠癌单克隆细胞株。方法:提取HCT116细胞总RNA,RT-PCR克隆PD-L1基因片段,将其重组至含有HA标签和Neo真核细胞筛选抗性的pcDNA3质粒载体上,转染HCT116细胞,倍比稀释8个细胞浓度至96孔板,G418筛选两周,挑取单细胞克隆,通过免疫印迹鉴定外源性PD-L1的表达;通过MTS、Transwell和软琼脂细胞克隆形成实验比较PD-L1低表达和高表达单克隆细胞株生长增殖、迁移和细胞克隆形成能力的差别;最后通过在两组细胞中过表达帽依赖性荧光素酶报告基因阐述造成上述表型的可能机制。结果:成功构建人PD-L1的真核表达载体,并筛选获得稳定高表达PD-L1的HCT116单克隆细胞株。功能研究初步证明PD-L1可促进细胞克隆形成能力、迁移和生长增殖。过表达PD-L1可使帽依赖性荧光素酶报告基因的表达上调约3~4倍,表明其机制部分为PD-L1可上调帽依赖性蛋白质翻译。结论:获得了PD-L1重组质粒载体及可用于后续功能研究的稳定高表达人PD-L1的HCT116单克隆细胞株。     

Pro-transforming growth factor-alpha processing in human colon carcinoma cells: Role of protein kinase C

The human colon cancer cell lines HCT 116 (poorly differentiated) and CEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-α). The secretion of TGF-α was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-α. Cells were metabolically labeled with ^3SS-cysteine and the formation of the mature 6 kDa TGF-α polypeptide from the 17 kDa pro-TGF-α precursor was determined. The conversion of pro-TGF-α was complete in 2–4 hr with the HCT 116 cells showing faster kinetics of TGF-α formation than GEO cells. HCT 116 cells secreted more TGF-α than GEO cells and the rate and extent of formation of TGF-α was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-α by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylac-etate 20-acetate (dPPA), stimulated the release of TGF-α into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-4bT1 specifically, our results suggest a potential role for PKC-β in the processing of pro-TGF-α by these 2 human colon carcinoma cell lines. © 1995 Wiley-Liss, Inc.