tetracycline and Colonic-Neoplasms

Topics: Administration, Oral; Aged; Clinical Trials as Topic; Colonic Neoplasms; Diverticulitis, Colonic; Female; Humans; Male; Middle Aged; Neomycin; Preoperative Care; Random Allocation; Sigmoid Diseases; Surgical Wound Dehiscence; Surgical Wound Infection; Tetracycline

A previous study has proven that cycle inhibiting factors (Cifs) inhibit Cullin E3 ubiquitin ligases, resulting in cell cycle arrest. More importantly, Cifs are also involved in cancer progression by deamidating Nedd8. Here we aimed to explore a novel insight into the treatment implications of Cifs in colon cancers by Tet-on system. The anticancer activity of Cif by doxycycline induction was investigated in the colon cell lines based upon Tet-On system. The expression of Cif in the colon cancer cells was determined by western blot. Furthermore, the cell viability and flow cytometry analysis were respectively performed to evaluate the cell proliferation and survival of colon cells. More importantly, the p21 and p27 levels were also evaluated after the induction of Cif with Tet-On system. Multiple clones of colon cancer cells for doxycycline-regulated Cif expression were constructed for maintenance purposes including HCT116 and SW480 cell lines. The result of western blot displayed good inducibility of expressing Cif in the cell lines. The clones with Cif preserved their transformed phenotype compared with the control group (clones with GFP or with Cif), in terms of the inhibition of cancer cell proliferation and survival. Furthermore, western blot analysis showed that p27 and p21 were accumulated in the clones with Cif, compared with the colon cancer cell lines with GFP or with Cif. Using the Tet-On system, we developed an efficient approach toward generation of colon cancer cells induced with Cif. These engineered colons tightly controlled Cif expression in vitro, which is a good inducible model system for cancer treatment.

Topics: Bacterial Proteins; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Clone Cells; Colonic Neoplasms; Doxycycline; Flow Cytometry; HCT116 Cells; Humans; Tetracycline

In normal colonic epithelium, the receptor tyrosine kinase, EphB2 interacts with ephrinB1 ligand to maintain the integrity and architecture of the colonic crypt. Loss of EphB2 is seen in most colorectal cancers and correlates with poor prognosis. In this study, we investigated the effects of two levels of EphB2 expression on cell migration and invasion in a colon cancer cell line and on the growth of tumour xenografts.. An EphB2-negative colon cancer cell line (LIM2405) was transfected with a full-length EphB2 cDNA in a vector designed to respond to the drug tetracycline. The effect of two levels of EphB2 expression on the ability of cells to migrate through a porous barrier in response to a chemo-attractant and to invade through artificial basement membranes was tested in vitro. Finally, the effects of two expression levels of EphB2 on tumour growth using an in vivo model of colonic tumour xenograft in a mouse model were assessed.. Expression of moderate levels of EphB2 significantly reduced the migration of tumour cells compared to control (p < 0.05, Kruskal-Wallis test). Expression of high levels of EphB2 further reduced migration of tumour cells (p < 0.05, Kruskal-Wallis test). Similarly, expression of EphB2 markedly reduces the invasive ability of tumour cells. The in vivo model of tumour growth showed that tumours with the highest level of EphB2 expression had a reduced risk of reaching a 10-mm size (defined event) compared with the control group (Cox regression, hazard ratio (HR) = 0.052, 95% CI 0.005-0.550; p = 0.014). Tumours derived from EphB2 expressing cells had a significantly reduced number of mitotic figures (p < 0.05) and an increased number of apoptotic cells (p < 0.05) compared to tumours from control cells.. Even a moderate level of EphB2 expression has effects on tumour cells which results in reduced migration and invasiveness and slows the growth of colonic tumour implants in an in vivo model.

Topics: Animals; Caspase 3; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Colonic Neoplasms; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Mice; Mitosis; Neoplasm Invasiveness; Receptor, EphB2; RNA, Messenger; Tetracycline; Xenograft Model Antitumor Assays

The recent finding that the 1p36.3 locus gene encodes an array of different p73 isoforms with apparently distinct and sometimes opposing cellular functions, might explain the difficulty in establishing the protein's role as tumor suppressor. Therefore we need to investigate the roles of each of these splicing variants in cellular functions when expressed alone or in combination with other family members, as well as the genetic background on which the proteins are expressed. We investigated, in two p53 null cell lines, the human SCLC line H1299 and a subline derived from the human colon carcinoma cell line HCT116 (HCT116/379.2), the effects of DeltaNp73alpha overexpression on cell growth and the response to anticancer treatment. We generated three different clones overexpressing DeltaNp73alpha under a tetracycline inducible promoter. Immunofluorescent staining and luciferase reporter assays confirmed that clones HCT116/DeltaNA and H1299/DeltaN7 and H1299/DeltaN11 did express a functional, nuclear localized DeltaNp73alpha protein. The stable overexpression of DeltaNp73alpha protein did not confer any cell growth advantage. Doubling time of clones overexpressing DeltaNp73alpha were comparable to counterparts not expressing it. Clonogenic assays showed that the cytotoxic activity of different DNA damaging agents, such as cDDP, UV light and doxorubicin, were comparable in clones expressing DeltaNp73 or not. The overall data argue against an oncogenic role for this isoform. These findings are independent of the p53 status since they overlap with those previously obtained by our group in HCT116 cell lines, wild type for p53.

Topics: Alternative Splicing; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; DNA-Binding Proteins; Humans; Nuclear Proteins; Promoter Regions, Genetic; Protein Isoforms; Sequence Deletion; Tetracycline; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

p53, a major sensor of DNA damage, is a transcription factor that, depending on its phosphorylation status, regulates the cell cycle, DNA repair, or apoptosis. The protein kinase C (PKC) family of isozymes is also implicated in cell cycle and programmed cell death (PCD) control and has recently been shown to influence p53 function. Using three human colon adenocarcinoma cell lines SW480, EB-1, and HCT116 that either lack p53 function and were engineered to express inducible wild-type p53 (wt p53), or that constitutively express wt p53, we show that phorbol ester-mediated PKC activation potentiates p53-induced PCD. Despite the effectiveness of PKC/p53 synergy in inducing SW480 tumor cell death, however, a fraction of the cells invariably survive. To address the putative mechanisms that underlie resistance to PKC/p53-induced cell death, we generated a phorbol 12-myristate 13-acetate/p53-resistant SW480 subline and compared the gene expression profile of resistant and parental cells by DNA microarray analysis. The results of these experiments show that PKC/p53-resistant cells express a higher level of several matrix metalloproteinases (MMP), including MMP-9, MMP-10, and MMP-12, and corresponding real-time PCR assays indicate that p53 is a negative regulator of MMP-9 gene expression. Using MMP inhibitors and MMP-specific small interfering RNA, we show that MMP function confers protection from PKC/p53-induced apoptosis and identify the protective MMPs as MMP-9 and MMP-10. Taken together, these observations provide evidence that MMPs are implicated in tumor cell resistance to the synergistic proapoptotic effect of PKC and p53.

Topics: Adenocarcinoma; Apoptosis; Caspases; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Humans; Intracellular Membranes; Matrix Metalloproteinase 10; Matrix Metalloproteinase 9; Membrane Potentials; Metalloendopeptidases; Mitochondria; Phosphatidylserines; Protein Kinase C; RNA, Small Interfering; Tetracycline; Tetradecanoylphorbol Acetate; Tumor Suppressor Protein p53

We report a case of a 61-year-old man who presented with fatigue, abdominal pain and hepatomegaly. Computed tomography (CT) of the abdomen showed hepatomegaly and multiple hepatic lesions highly suggestive of metastatic diseases. Due to the endoscopic finding of colon ulcer, colon cancer with liver metastases was suspected. Biochemically a slight increase of transaminases, alkaline phosphatase and gammaglutamyl transpeptidase were present; alpha-fetoprotein, carcinoembryogenic antigen and carbohydrate 19-9 antigen serum levels were normal. Laboratory and instrumental investigations, including colon and liver biopsies revealed no signs of malignancy. In the light of spontaneous improvement of symptoms and CT findings, his personal history was reevaluated revealing direct contact with pigs and their tissues. Diagnosis of leptospirosis was considered and confirmed by detection of an elevated titer of antibodies to leptospira. After two mo, biochemical data, CT and colonoscopy were totally normal.

Topics: Colonic Neoplasms; Colonoscopy; Contrast Media; Diagnosis, Differential; Gram-Negative Bacteria; Humans; Leptospirosis; Liver Function Tests; Liver Neoplasms; Male; Middle Aged; Tetracycline; Tomography, X-Ray Computed

To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene.. HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein.. Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably.. High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.

Topics: Blotting, Western; Cell Division; Colonic Neoplasms; Flow Cytometry; G2 Phase; GADD45 Proteins; HCT116 Cells; Humans; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Mitosis; Proteins; Tetracycline

Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for maintenance of growth and tumorigenicity of several tumor types. We have previously shown successful therapy in a lung cancer xenograft model using an adenovirus expressing antisense IGF-Ir. In this study, we sought to better dissect the mechanism and develop potentially more effective IGF-Ir-targeted therapeutics by developing and testing tetracycline-regulated and recombinant adenoviruses expressing dominant negative receptors.. Truncated IGF-I receptors (IGF-Ir/tf; 482 and 950 amino acids long, respectively [IGF-Ir/482st and IGF-Ir/950st]) were cloned into tetracycline-regulated vectors and recombinant adenoviruses and then studied in colorectal cancer cells. We assessed the effect of IGF-Ir/tf on signaling blockade, colony formation, stress response (serum starvation and heat), chemotherapy-induced apoptosis, and in vivo therapeutic efficacy in xenografts.. Activation of IGF-Ir/tf expression by withdrawal of tetracycline suppressed tumorigenicity both in vitro and in vivo and up-regulated stressor-induced apoptosis. It effectively blocked both IGF-I- and IGF-II-induced activation of Akt-1. IGF-Ir/tf expression increased chemotherapy-induced apoptosis, and this combination therapy was very effective against tumors in mice. These findings were confirmed in a therapy model against established tumors using adenoviruses expressing IGF-Ir/tf. Moreover, IGF-Ir/482st was more effective than IGF-Ir/950st because of its bystander effect.. Anti-tumor activity of IGF-Ir/tf is mediated through inhibition of Akt-1 and enhances the efficacy of chemotherapy. Adenovirus IGF-Ir/482st may be a useful anticancer therapeutic for colorectal carcinoma.

Topics: Adenoviridae; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Blood Proteins; Carcinogenicity Tests; Cell Division; Colonic Neoplasms; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; Genetic Therapy; Heat-Shock Response; HT29 Cells; Humans; Mice; Mice, Nude; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Tetracycline; Transfection

Clinical studies suggest that carcinoembryonic antigen (CEA) is associated with metastatic progression of colon cancer. However, the biological function of CEA is not well understood. We have established an approach that allows studying of CEA function within the intact pathophysiological context of human colon cancer cells.. We expressed CEA-targeted ribozymes under control of a tet-off promoter system in human HT29 colon cancer cells. This approach allows regulation of CEA levels on the mRNA and protein level by 50% and enables screening analysis of CEA-mediated changes of gene expression by cDNA microarray analysis.. Comprehensive analysis of 273 genes revealed that CEA affects expression of various groups of cancer-related genes, in particular cell cycle and apoptotic genes. Although cell cycle gene expression showed a balanced bidirectional dysregulation, apoptotic genes were unidirectionally down-regulated by CEA. In parallel phenotypic studies, CEA did not affect cell cycle or proliferation rate. However, CEA significantly protected HT29 cells from undergoing apoptosis under various conditions, including confluent growth, UV light, IFN-gamma treatment, and treatment with 5-fluorouracil.. Our study suggests that CEA has an important regulatory role in apoptosis, and we propose that CEA is a survival factor for colon cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Northern; Carcinoembryonic Antigen; Cell Count; Cell Cycle; Cell Division; Colonic Neoplasms; Fluorouracil; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Interferon-gamma; Plasmids; RNA, Catalytic; RNA, Messenger; Tetracycline; Trans-Activators; Transfection

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death, and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. To help understand these mechanisms we have established stable derivatives of the K12 rat colon carcinoma cell line that express bcl-XS in a tetracycline-regulated manner, using an autoregulatory retroviral cassette. When bcl-XS expression is induced, we observe two phenotypic responses. A small fraction of cells appear to undergo spontaneous apoptosis while the majority of cells undergo a form of cytostasis. In the latter case, the cells stop dividing (or divide a limited number of times at a retarded rate) and swell to many times their original size. These cells can take on a ghostlike appearance and subsequently detach from the culture plates and die or they may remain intact in a hindered state of proliferation. Doubling times were calculated to be 31.4 h in the presence of tetracycline and 50.4 h without tetracycline, bcl-XS expression also causes dramatic alterations in the cell cycle distribution of K12 cells manifesting as a substantial decrease (approximately 50%) in the fraction of S phase cells with a concomitant increase in the G1 population. Continuous expression of bcl-XS, at levels approximately equal to that of bcl-XL, decreased the viability of K12 cells as demonstrated by a log decline in clonogenic survival. This decrease occurred without considerable apoptosis or a compensatory increase in the level of bcl-XL. None of these phenotypes were present in control cells expressing beta-galactosidase in a similar retroviral cassette. These observations demonstrate that bcl-XS can have substantial cytokinetic effects under circumstances that produce relatively little apoptosis.

Topics: Animals; bcl-X Protein; Cell Cycle; Cell Division; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Gene Expression Regulation; Lac Operon; Proto-Oncogene Proteins c-bcl-2; Rats; Tetracycline; Time Factors; Tumor Cells, Cultured

We have previously shown that the human ETS1 protein (p51-ETS1), when ectopically expressed in colon cancer cell lines, is able to reduce its tumorigenicity without affecting its growth properties. To understand the mechanism of tumor reduction, we have expressed two different forms of ETS1 in colon cancer cell lines. Data presented in this paper indicate that the naturally occurring spliced variant protein, p42-ETS1, lacking the region encoded by ETS1 exon VII, represses the tumorigenicity, while p51-ETS1 reduces the tumorigenicity. Repression of tumorigenicity mediated by p42-ETS1 appears to be caused by its ability to induce apoptosis in epithelial cancer cells. This work can have profound medical significance in that it may open up new insights into the potential role of the p42-ETS1 in the induction of apoptosis in epithelial cell cancers and may provide a rationale for its use for potential gene therapy experiments to initiate cell death in cancer cells.

Topics: Animals; Apoptosis; Cell Nucleus; Colonic Neoplasms; Humans; Mice; Mice, Nude; Protein Synthesis Inhibitors; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Tetracycline; Transcription Factors; Transfection; Tumor Cells, Cultured

Transforming growth factor beta (TGF-beta) promotes tumor progression in some model systems including human breast cancer cells. In this study, we report that human breast cancer cell lines express reduced amounts of TGF-beta type III receptor (RIII) when compared with untransformed human mammary epithelial cells. Consequently, we examined whether expression of RIII in human breast cancer MDA-MB-231 cells could reduce TGF-beta's tumor promoting activity by sequestering active TGF-beta isoforms produced by the cells. A tetracycline-repressible human RIII expression vector was stably transfected into the cell line. RIII expression in a pool of transfected clones and a single clone was found to be reversibly repressed by tetracycline treatment. Expression of RIII reduced the amount of active TGF-beta1 and TGF-beta2 in the conditioned medium. The medium conditioned by control cells showed a significantly higher growth inhibitory effect than that conditioned by RIII-transfected cells on the growth of the mink lung epithelial CCL64 cells. A conditioned medium collected from RIII-transfected cells treated with tetracycline significantly increased its growth inhibitory activity to that of control cells. Expression of RIII also reduced tumor incidence and growth rate in two separate experiments when the cells were inoculated in athymic nude mice. Treatment of the mice with tetracycline repressed RIII expression in the tumors generated by RIII-transfected cells and increased tumor incidence and growth rate. These results suggest that TGF-beta RIII can reduce tumorigenicity of MDA-MB-231 cells apparently by sequestering TGF-beta isoforms produced by these cells.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lung; Mice; Mice, Nude; Mink; Proteoglycans; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Tetracycline; Transcription, Genetic; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Transforming growth factor beta (TGFbeta) type I (RI) and type II (RII) receptors are essential for TGFbeta signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFbeta and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFbeta. Hence, the role of RI as a limiting factor for TGFbeta sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFbeta1 binding to RI and resulted in increased growth inhibition by exogenous TGFbeta1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFbeta1 binding to RII, growth inhibition by exogenous TGFbeta1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFbeta in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFbeta1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFbeta activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFbeta, as well as TGFbeta autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.

Topics: Activin Receptors, Type I; Animals; Antigens, CD; Cell Division; Cell Line; Cloning, Molecular; Colonic Neoplasms; DNA Replication; Fibronectins; Humans; Integrin alpha5; Mice; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Tetracycline; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Colonic Neoplasms; Fluorescence; Humans; Tetracycline

Topics: Aged; Bacitracin; Colectomy; Colon; Colonic Diseases; Colonic Neoplasms; Colostomy; Drug Therapy, Combination; Humans; Neomycin; Peritonitis; Postoperative Complications; Premedication; Rectal Diseases; Rectal Neoplasms; Rectum; Sepsis; Surgical Wound Infection; Tetracycline

Topics: Adolescent; Adult; Aged; Anti-Bacterial Agents; Bacteroides; Bacteroides Infections; Breast Neoplasms; Child; Colonic Neoplasms; Female; Genital Neoplasms, Female; Hospitals, Special; Humans; Leukemia; Lymphoma; Male; Middle Aged; Neoplasms; Pressure Ulcer; Sepsis; Surgical Wound Infection; Tetracycline; Time Factors

Topics: Adenocarcinoma; Carcinoma; Colonic Neoplasms; Diagnosis, Differential; Diverticulitis, Colonic; Fluorescence; Humans; Microscopy, Fluorescence; Prognosis; Rectal Neoplasms; Sigmoid Neoplasms; Stomach Neoplasms; Tetracycline

Topics: Colonic Neoplasms; Colostomy; Drug Therapy; Humans; Ileostomy; Kanamycin; Neomycin; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasms; Postoperative Complications; Preoperative Care; Rectal Neoplasms; Rectum; Sulfonamides; Surgical Procedures, Operative; Sutures; Tetracycline

Topics: Anesthesia; Atropine; Barbiturates; Chemical and Drug Induced Liver Injury; Colonic Neoplasms; Halothane; Hepatitis; Massive Hepatic Necrosis; Necrosis; Neomycin; Phenazocine; Postoperative Complications; Promethazine; Sigmoidoscopy; Surgical Procedures, Operative; Tetracycline; Toxicology

Topics: Colon, Sigmoid; Colonic Neoplasms; Colostomy; Diagnosis, Differential; Diverticulitis; Diverticulitis, Colonic; Fistula; Geriatrics; Humans; Intestinal Obstruction; Mortality; Neomycin; Peritonitis; Postoperative Complications; Preoperative Care; Rectum; Statistics as Topic; Sulfathiazoles; Surgical Procedures, Operative; Surgical Wound Infection; Tetracycline

Topics: Anti-Bacterial Agents; Colonic Neoplasms; Diagnosis; Enteritis; Humans; Neomycin; Postoperative Complications; Staphylococcal Infections; Staphylococcus; Tetracycline