Rat Colon Carcinoma Cell Line Research Articles

Intraoperative versus Early Postoperative Intraperitoneal Chemotherapy after Cytoreduction for Colorectal Peritoneal Carcinomatosis: an Experimental Study

Perioperative intraperitoneal chemotherapy is used as an adjunct to cytoreductive surgery (CS) for peritoneal carcinomatosis (PC) in order to prolong survival. Worldwide, hyperthermic intraperitoneal chemotherapy (HIPEC), early postoperative intraperitoneal chemotherapy (EPIC), and combinations of the two are used. It remains unclear which regimen is most beneficial. The rat colon carcinoma cell line CC-531 was injected into the peritoneal cavity of 80 WAG/Rij rats to induce PC. Animals were randomized into four treatment groups (n = 20): CS only, CS followed by HIPEC (mitomycin 35 mg/m(2) at 41.5°C), CS followed by EPIC during 5 days (i.p. injection of mitomycin on day 1 and 5-fluorouracil on days 2-5), and CS followed by HIPEC plus EPIC. Primary outcome was survival. In rats treated with CS only, median survival was 53 days (95% confidence interval (CI) 49-57 days). In rats treated with CS followed by HIPEC, survival was significantly (P = 0.001) increased (median survival 94 days, 95% CI 51-137 days). In the group treated with EPIC after CS, 12 out of 20 rats were still alive at the end of the experiment (P < 0.001 as compared with CS only). In the group receiving both treatments, 11 rats died of toxicity, and therefore this group was not included in the survival analysis. Both EPIC and HIPEC were effective in prolonging survival. The beneficial effect of EPIC on survival seemed to be more pronounced than that of HIPEC. Further research is indicated to evaluate and compare the possible benefits and adverse effects associated with both treatments.

Hyperthermia and Intraperitoneal Chemotherapy for the Treatment of Peritoneal Carcinomatosis

Hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C can improve survival if used as an adjunct to cytoreductive surgery (CS) for treatment of peritoneal carcinomatosis (PC). It remains unclear if both hyperthermia and chemotherapy are essential for the reported survival benefit. Eighty WAG/Rij rats were inoculated intraperitoneally with the rat colon carcinoma cell line CC-531. Animals were randomly assigned to 1 of the 4 treatment groups (n = 20): CS only, CS followed by HIPEC (mitomycin 35 mg/m(2) at 41°C), CS followed by intraperitoneal mitomycin perfusion at 37°C, CS followed by intraperitoneal saline perfusion at 41°C. Survival was the primary outcome with a maximum follow up of 126 days. Median survival was 62 days in rats treated with CS only and 57 days in rats treated with CS followed by hyperthermic saline perfusion. Rats receiving HIPEC had a median survival of 121 days (P = 0.022 when compared with CS only). In the group treated with chemotherapy at 37°C, 13 of 20 animals were still alive at the end of the experiment so median survival was not reached. (CS vs. IPEC: P = 0.002, hazard ratio 0.36, 95% CI 0.19-0.69) Rats treated with hyperthermic saline perfusion did not have an increased survival as compared with CS only. The effectiveness of intraoperative intraperitoneal perfusion after CS is highly dependent on the presence of chemotherapeutic agents in the perfusate but not on hyperthermia. The need to include hyperthermia in the adjuvant intraoperative treatment after CS for PC should be further investigated.

In VitroandIn VivoStudies of Boron Neutron Capture Therapy: Boron Uptake/Washout and Cell Death

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal-neutron irradiation of cells enriched with (10)B, which produces α particles and (7)Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron-enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron-loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation-induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.

Establishment of conditionally immortalized epithelial cell lines from the intestinal tissue of adult normal and transgenic mice

It has proved to be impossible to culture epithelial cells from the gastrointestinal tract of adult animals. Researchers have had to use either cell lines derived from newborn rat small intestine or colon carcinoma cell lines that have retained some of the properties of the gastrointestinal mucosa. We have described a method for establishing conditionally immortalized cell lines from the stomach, small intestine, colon, pancreas, and liver from tissue obtained from a transgenic mouse strain carrying a temperature-sensitive mutant of the SV40 large T gene (the "Immortomouse"). This immortalizing gene has proved to be useful for establishing cell lines from a number of transgenic mice following crossbreeding of the Immortomouse with the transgenic mouse of interest. These cell lines are being used in numerous studies. In this review we describe the methods for developing such lines and list the range of cell lines that have been developed from colon, small intestine, stomach, liver, and pancreas of a number of transgenic mice.

Disrupted Expression of CXCL5 in Colorectal Cancer Is Associated with Rapid Tumor Formation in Rats and Poor Prognosis in Patients

We isolated a subline (CC531M) from the CC531S rat colon carcinoma cell line, which grows and metastasizes much more rapidly than CC531S. We found, using RNA expression profiling, that one of the major changes in the CC531M cell line was a 5.8-fold reduction of the chemokine CXCL5. The purpose of this study was to determine the effect of CXCL5 expression on colorectal tumor growth and metastasis. CC531 clones were generated with either knockdown or restored expression of CXCL5. These clones were inoculated in the liver of rats. In addition, in two independent cohorts of colorectal cancer patients, the level of CXCL5 expression was determined and associated to clinical variables. Knockdown of CXCL5 expression in CC531S resulted in rapid tumor growth and increased number of metastasis, whereas restored expression of CXCL5 in CC531M resulted in a return of the "mild" tumor growth pattern of the parental cell line CC531S. In vitro, no difference was found in proliferation rate between clones with either high or low expression of CXCL5, suggesting that environmental interactions directed by CXCL5 determine tumor outgrowth. Finally, the importance of our findings was established for patients with colorectal cancer. We found that low expression of CXCL5 was significantly associated with poor prognosis for colorectal cancer patients. CXCL5 showed a trend (P = 0.05) for a positive correlation with intratumoral CD8(+) T-cell infiltration, suggesting a possible explanation for the observed poorer prognosis. Our results show that CXCL5 is important in growth and development of colorectal cancer, implicating a future role in both cancer therapy and diagnosis.

Anti-β1 Integrin Antibody Reduces Surgery-Induced Adhesion of Colon Carcinoma Cells to Traumatized Peritoneal Surfaces

To study the mechanisms behind surgery-induced augmentation of tumor outgrowth. Surgery provides the best chance of cure for most primary intra-abdominal carcinomas. Effective treatment is however relatively frequent complicated by peritoneal recurrences, which often originate from free-floating intraperitoneal tumor cells that implant on peritoneal surfaces. We previously reported that surgical trauma promotes development of peritoneal metastases. Evaluation of adhesion of CC531s rat colon carcinoma cell line intraperitoneally after laparotomy using in vivo, ex vivo, and in vitro models. Also, human ex vivo models were used to study peritoneal tumor cell adhesion. Peritoneal imprints of operated rats showed that direct damaging of the peritoneum resulted in enhanced adhesion of rat CC531 colon carcinoma cells to submesothelial extracellular matrix (ECM) proteins in vivo, which was confirmed by electron microscopy. Additionally, the inflammatory reaction of the peritoneal cavity led to retraction of mesothelial cells, hereby also exposing ECM at peritoneal surfaces that had not been traumatized directly. Furthermore, we demonstrated that beta1 integrin subunits represented the primary mediators involved in adherence to either isolated ECM components or excised traumatized rat and human peritoneum. Importantly, incubation of CC531s cells with anti-beta1 integrin antibodies resulted in a significant decrease of tumor cell adhesion in vivo. Surgical trauma results in exposure of ECM at directly and nondirectly damaged peritoneal surfaces, leading to increased beta1 integrin-dependent tumor cell adhesion. Perioperative therapies, which aim to block beta1 integrin subunits, might therefore serve as new clinical tools for the prevention of peritoneal recurrences.

965. A Novel Approach for Gene Transduction with Adenovirus/Fibrin Glue System

Introduction: The major cause of local recurrence after initial surgery for hepatic malignancy is residual carcinoma macroscopically or microscopically at the surgical site. Although many surgical devices and skills have developed, the recurrence rate of hepatic malignancy is still high. At present, for those patients with recurrent disease, adjuvant therapies such as chemotherapy, radiation therapy and immunotherapy are combined. However, in such cases, severe side-effects make the patient's QOL (quality of life) considerably worse. Therefore, a novel approach for residual carcinoma at the time of surgery is urgently needed. In this study we proposed the use of a novel gene therapy for residual carcinoma of the liver. Purpose: The fibrin glue system (FGS) consists of liquid forms of fibrinogen and thrombin and is used widely in surgical operation as it has a sealing effect and promotes tissue adhesion at the surgically resected site. In this study, we proposed the efficacy and safety of loco-regional gene transfer using adenoviral vector mixed with FGS to prevent local recurrence of liver carcinoma. We demonstrated that the adenoviral vector containing the E.coli LacZ gene (AxCALacZ) was delivered to the liver of rats by FGS. Materials and Methods: Experiment 1 in vitro Rat colon carcinoma cell line, RCN-9 was incubated on a membrane containing multiple 0.02μm-sized holes. Various concentrations of the adenoviral vector mixed with FGS (AxCALacZ/FGS) were applied on the RCN-9 and incubated for 48 hours. The optimum efficiency of transduction was evaluated by X-Gal staining and NIH imaging. Experiment 2 in vitro To examine the survival period of adenoviral vector in FGS, AxCALacZ/FGS put on the monolayer cultured RCN9 after being left for various periods. Transgene expression was evaluated by β-gal assay. The β-gal activity was measured photometrically using the Promega β-gal enzyme assay kit according to the manufacture's instructions (Promega, Madison, USA). Experiment 3 in vivo 8-week-old male F344 rats underwent a partial hepatectomy. AxCALacZ/FGS was applied on the cut surface of the dissected liver and the rats were kept for 48 hours under normal conditions. AxLacZ/PBS was treated as the control study. The transducted and non-transducted parts of the liver were extirpated, stained with X-gal liquid and β-gal was measured to compare the efficacy of transduction in each organ. Results: Experiment 1 significantly more transgene expression was shown by the X-gal staining and NIH imaging than 1MOI. Experiment 2 LacZ expression revealed infected RCN9 when AxCALacZ/FGS was left for a period of less than 96 hours. Experiment 3 AxCALacZ/FGS transgene expression was shown at the treated and non-treated part of the liver more than AxCALacZ/PBS expression. Discussion and conclusion: It was demonstrated that adenoviral vector survives and being stable for enough time to transduct in FGS. AxCALacZ/FGS can transduct the target gene. Furthermore, the transduction efficiency was comparable to those of existing methods. In the future, FGS will be studied with an adenoviral vector containing the gene which enhances antitumor immunity for preventing recurrence loco-regionally site as well as its original tissue adhesive purpose.

Cytotoxic reactions of CC531s towards liver sinusoidal endothelial cells: a microscopical study

Colorectal cancer cells can induce apoptosis in cells of various tissues [1]. Apoptosis can be induced by a number of factors such as Fas inducing apoptosis through the Fas/FasL pathway; other factors involve the TRAIL pathway and TNF. It is known that metastasizing colon cancer cells express more FasL then primary carcinoma cells [2]. We investigated whether a rat colon carcinoma cell line CC531s could induce apoptosis in liver sinusoidal endothelial cells (LSECs). LSECs and CC531s were co-cultured for 18 hrs and cells were visualized by SEM and TEM. Apoptosis was visualized by markers such as Hoechst and Propidium iodide. Furthermore, cells were recorded by time lapse video microscopy with and without an antagonistic antibody for FasL.

Butyrate reduces liver metastasis of rat colon carcinoma cells in vivo and resistance to oxidative stress in vitro

Injection of the rat colon carcinoma cell line CC531 into spleen of syngeneic rats results in considerable amounts of liver metastases within 14 days. We investigated whether preincubation of the cells with butyrate reduced their metastatic ability in vivo and whether this was accompanied by reduction in related properties such as secretion of metalloproteinases and their ability to withstand oxidative stress. Butyrate incubation reduced cell growth rate and initiated apoptosis in a dose- and time-related manner, but proliferation was retrieved when cultivation was continued in medium without butyrate. Splenic injection of butyrate treated, proliferating cells resulted in significantly reduced amounts of tumor mass compared to untreated cells. The butyrate treated cells were more susceptible to oxidative stress than control cells, as demonstrated by increased number of apoptotic cells and reduced cell growth after exposure to menadione. A reduction in cellular glutathione was found after prolonged incubation with butyrate. Butyrate appeared not to alter the secretion of active metalloproteinases from the cells although an apparent increase in proforms was demonstrated. Neither did butyrate alter the synthesis of metalloproteinase inhibitors. Lastly, a reduced adhesion of the tumor cells to collagen coated matrix was found after butyrate treatment. Thus, the inhibitory effects of butyrate on tumor malignancy are caused by a diversity of mechanisms.

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.

Immunological aspects of photodynamic therapy of liver tumors in a rat model for colorectal cancer.

We have investigated tumor immunological effects of photodynamic therapy (PDT) of liver metastases. Livers of Wag/Rij rats were inoculated with three tumors of a syngeneic rat colon carcinoma cell line, CC531. One tumor in each rat was illuminated, with or without previous administration of the photosensitizer metatetrahydroxyphenylchlorin (mTHPC). PDT was effective in causing necrosis of tumors, but it did not affect the growth rate of nearby, nonilluminated tumors in the liver. Immunological staining of tumors showed natural killer (NK) cells to be significantly lower in PDT-treated tumors than in control tumors (P < 0.05). T cells in PDT-treated tumors and in their margins were lower than in tumors that received only sensitizer or only illumination (P = 0.015) at day 2 after treatment but reappeared at the tumor margins from day 7 after treatment. For macrophages, a similar pattern was found. NK cells, T cells or macrophages in nonilluminated tumors in mTHPC-treated rats did not increase significantly when compared with tumors in rats without mTHPC treatment. These findings indicated that no antitumor effect of a systemic immune response was present, as measured by the effect of PDT on growth of distant tumors and the number of T lymphocytes, NK cells and macrophages in these tumors.

Rat colon carcinoma cells that survived systemic immune surveillance are less sensitive to NK-cell mediated apoptosis.

In order to form distant metastases, cells from the primary tumor have to detach, enter the blood- or lymph-compartment and escape immune surveillance. Here, we describe the selection of rat colon carcinoma cell lines (CC531s-m1 and CC531s-m2) that escaped from systemic immune surveillance; CC531s cells were injected into the v. jugularis of Wag/Rij rats, after three weeks the lung tumors were isolated, the tumor cells were cultured, characterized and injected again. The m1- and m2-cell lines were less susceptible for killing by syngeneic NK cells. Further characterization of this cell line showed a decreased sensitivity towards TRAIL- and CD95L-, but not to granzyme B-mediated apoptosis. In the m1- and m2-cells log-phase growth started earlier as compared to the parental cell line, whereas no changes were found in anchorage-dependent or anchorage-independent growth. After subcapsular injection of the m2-cell line into the liver of rats much more lung metastases were formed in comparison to injection of the parental cell line. In conclusion, the results suggest that the resistance of the m1- and m2-cells to NK cell-mediated apoptosis was associated with their capability to survive systemic immune surveillance and form metastases in vivo.

Expression of hyaluronan synthase 2 or hyaluronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell tumors.

Advanced colorectal cancers are often associated with elevated amounts of hyaluronan. To investigate the importance of hyaluronan in colon carcinoma tumor progression, we have expressed by stable transfection hyaluronan synthase 2 (Has2) and hyaluronidase 1 (Hyal1) in the rat colon carcinoma cell line, PROb. We found that hyaluronan overproduction led to a higher growth rate of tumor cells in vitro, and to a faster development of transplantable tumors in syngeneic rats, compared to the mock-transfectants. Has2 transfected PROb cells gave rise to tumors that were significantly less vascularized, but had a significantly larger viable tumor fraction compared to tumors generated from mock-transfectants. In contrast, Hyal1 overexpression suppressed the growth rate of tumor cells both in vitro and in vivo. Moreover, tumors derived from Hyal1-transfected cells had a significantly larger necrotic area than tumors derived from mock- and Has2-transfectants. Our study demonstrates that Has2 overproduction promotes tumorigenicity, whereas Hyal1 overexpression suppresses tumorigenicity in an experimental model for colon carcinoma.

Activation of the γ-glutamyltransferase promoter 2 in the rat colon carcinoma cell line CC531 by histone deacetylase inhibitors is mediated through the Sp1 binding motif

The single-copy gene for rat γ-glutamyltransferase (GGT) encodes at least seven distinct mRNAs that differ in their 5′-untranslated regions only. Tissue- and developmental-specific expression of GGT is partly achieved by the presence of many transcription factor-binding sites in the promoters of this gene. In an earlier study we found that GGT mRNAs II and IV levels were increased upon butyrate-induced differentiation of the rat colon carcinoma cell line CC531. The mechanism for this butyrate-induced upregulation remains unknown, but may result from altered promoter activity as butyrate is a known histone deacetylase inhibitor. In the present study, we show by transient transfection studies that butyrate enhanced the expression of the luciferase reporter gene driven by the rat GGT promoter 2 (P2). Trichostatine A (TSA), another histone deacetylase inhibitor, also enhanced transcription from this promoter. The role of the transcription factor site Sp1 in butyrate- or TSA-induced activation of the GGT P2 was examined as Sp1 has been previously shown to play a central role in the transcriptional activation of other genes during butyrate and TSA stimulation. A triple sequence-motif of this isolated Sp1 site linked to a minimal promoter was able to mediate butyrate- and TSA-induced expression of the luciferase reporter gene, while no effect was measured using the minimal promoter alone. Deleting the Sp1 site in the context of the rat GGT P2 strongly reduced the basal transcription activity and abrogated butyrate- and TSA-induced activation of the mutated promoter. These results suggest that butyrate- or TSA-induced activation of the rat GGT P2 can be mediated by a Sp1 binding motif.

Distinct Intracellular Signaling in Tumor Necrosis Factor-related Apoptosis-inducing Ligand- and CD95 Ligand-mediated Apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.

Prerequisites for effective adenovirus mediated gene therapy of colorectal liver metastases in the rat using an intracellular neutralizing antibody fragment to p21-Ras.

Ras mutations are present in 40–50% of colorectal cancers. Inactivating this oncogene may therefore reduce proliferation capacity. In order to target ras we studied the transduction efficacy and anti tumour activity of an adenoviral vector expressing an intracellular, neutralizing single chain antibody to p21-ras (Y28). In in vitro studies transfection levels of the K-ras mutated rat colon carcinoma cell line CC531 were studied using the LacZ marker gene. In our in vivo liver metastases model different routes of administration were evaluated to determine which regimen resulted in the best transfection levels and tumour responses: intravenous injection, intratumoural injection, isolated liver perfusion, or hepatic artery infusion. CC531 cells are readily transfected in vitro, resulting in significant inhibition of tumour cell proliferation by the Y28 construct. Intravenous injection did not result in any measurable transfection. Intratumoural injection resulted only in the transfection of tumour cells along the needle track. IHP as well as single HAI achieved low transfection levels of tumour tissue. Expression of Y28 was demonstrated in tumours after IT injection, HAI and IHP. Whereas, repeated HAI's clearly achieved expression in and around tumour associated vessels. Only five times repeated HAI's with Y28 resulted in a tumour response: in all animals tumour growth was inhibited, and in three rats out of eight a complete regression of the liver tumours was observed.British Journal of Cancer (2002) 86, 436–442. DOI: 10.1038/sj/bjc/6600089 www.bjcancer.com© 2002 The Cancer Research Campaign

A rat model to study the role of STn antigen in colon cancer.

Expression of the mucin-associated sialyl-Tn (STn) antigen has been associated with a decreased survival in patients with colorectal, gastric, and ovarian cancer. To better understand the role of STn antigen in tumor biology, we developed STn(+) (called LP) and STn(-) (called LN) clonal cell lines from a parental metastatic rat colon carcinoma cell line (LMCR). Both derivative cell lines exhibited identical proliferation rates in vitro. LP cells strongly expressed STn antigen both in vitro and in vivo, and were poorly tumorigenic when given to syngeneic rats. LN cells did not express STn antigen in vitro, but as in vivo tumors these cells rapidly acquired STn expression, readily formed tumors, and were highly lethal. When rats were given an otherwise lethal inoculum of i.p. LN cells, pre-immunization with synthetic STn antigen conjugated to keyhole limpet hemocyanin (STn-KLH) resulted in a 60% survival rate. When LN cells were injected subcutaneously in the presence of STn-KLH-sensitized lymphocytes, tumor growth was decreased. Distribution of STn antigen in normal organs of host rats is quite similar to that of humans. This model mimics human disease and should facilitate studies of mucin-associated antigens in tumor biology and the development of immunotherapeutic agents based on mucin-related antigens.

Vaccination par thérapie génique suicide contre un modèle de métastases hépatiques de cancer colique chez le rat

Suicide gene therapy consists of transferring into tumor cells a viral or bacterial gene encoding for an enzyme which converts a non-toxic product into a lethal drug.Study aim: To analyze the therapeutic potential of vaccination with tumor cells expressing the bacterial cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) treatment in a rat liver metastasis model.Material and method: We used a rat colon carcinoma cell line which, after subcapsular or intraportal injection in syngenic animals, generates single or multiple experimental liver metastases, respectively. We have shown that introduction of a vector expressing the CD gene in this colon carcinoma cell line results in 5-FC sensitivity (PRObCD).Results: Intrahepatic subcapsular injection of PRObCD tumor cells, followed by 5-FC treatment, induces total regression of a wild-type tumor pre-established in the contralateral liver lobe in 45% of animals with a 96% decrease in mean volume (p < 0.0001), demonstrating the existence of a distant bystander effect. This vaccination significantly increased the survival of rats with single (log-rank p < 0.0001) or multiple (log-rank p = 0.01) liver metastasisConclusions: These results suggest that suicide gene-modified tumor cells can act as potent therapeutic vaccines against liver metastasis from colon carcinoma.

Endothelin receptor blockade potentiates FasL-induced apoptosis in rat colon carcinoma cells.

Imbalanced proliferation and apoptosis is important in tumor progression. Endothelin (ET)-1, a 21-amino-acid peptide with vasoconstricting and mitogenic activities, has been shown to be involved in the regulation of apoptosis. Progressive and regressive rat colon (PROb and REGb cells) carcinoma cell lines express the components of the ET-1 system (preproET-1, ET-converting enzyme and ET-receptors) and secrete ET-1. These cells also express the Fas(APO-1, CD95)/FasL system, but are resistant to FasL-induced apoptosis. We thus addressed the role of ET-1 in FasL-dependent cell death. Bosentan, a mixed ET(A)/ET(B) receptor antagonist, potentiated FasL-induced apoptosis in these cells. At low concentrations (10(-13) to 10(-10) M), ET-1 dose-dependently reversed bosentan-induced apoptosis. Bosentan sensitization to FasL-induced apoptosis was not mediated by increased expression of Fas receptor and was blocked by the caspase inhibitor zVAD-fmk. The specific inhibition of enzymes involved in ceramide production did not restore survival of cells exposed to FasL and bosentan. Our results suggest that ET-1 is a survival factor able to protect in vitro colon carcinoma cells against FasL-induced apoptosis.

Regression of experimental liver tumor after distant intra-hepatic injection of cytosine deaminase-expressing tumor cells and 5-fluorocytosine treatment.

Cytosine deaminase (CD) gene of E. coli converts the non-toxic compound 5-fluorocytosine (5-FC) into 5-fluorouracil. We have introduced a vector expressing the CD gene in a rat colon carcinoma cell line. Expression of the CD gene confers 5-FC sensitivity to these cells in vitro and in vivo. In a bifocal model consisting in a simultaneous engrafment of a CD+ tumor on one lobe of the liver and a wild-type parental tumor on the opposite lobe, treatment with 5-FC results in regression of both type of tumors, indicating the existence of a distant bystander effect.