Cultured Carcinoma Cell Lines Research Articles

DNAX-activating protein 10 co-stimulation enhances the anti-tumor efficacy of chimeric antigen receptor T cells

ABSTRACT Chimeric antigen receptor (CAR) T cell immunotherapies have shown remarkable efficacy in treating multiple types of hematological malignancies but are not sufficiently effective at treating solid tumors. NKG2D is a strong activating receptor for NK cells and a co-stimulatory receptor for T cells. NKG2D signal transduction depends on DNAX-activating protein 10 (DAP10). Here, we introduced the cytoplasmic domain of DAP10 into the second-generation CARs M28z and G28z to generate M28z10 and G28z10, which target mesothelin (MSLN) and glypican 3 (GPC3), respectively. T cells expressing M28z10 or G28z10 showed enhanced and prolonged effector function against MSLN+ lung cancer or GPC3+ hepatocellular carcinoma cell lines in culture and secreted elevated levels of cytokines, including IL-2, IFN-γ, granzyme B, and GM-CSF. In addition, M28z10 CAR-T cells showed greater anti-tumor activity than those expressing M28z in both A549 cell line xenografts and human lung cancer patient-derived xenografts (PDX). Similarly, G28z10 exhibited higher efficacy in causing tumor regression than did G28z in hepatocellular carcinoma PDX. Therefore, our results show that DAP10 signaling contributes to the function of CAR-T cells in both lung cancer and hepatocellular carcinoma and can enhance the efficacy of CAR-T cells.

CFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition.

Epithelial cancers (carcinomas) comprise the top four causes of cancer-related deaths in the United States. While overall survival has been steadily improving, therapy-resistant disease continues to present a major therapeutic challenge. Carcinomas often exploit the normal developmental program, epithelial-to-mesenchymal transition (EMT), to gain a mesenchymal phenotype associated with increased invasiveness and resistance to apoptosis. We have previously shown that an isoxazole-based small molecule, ML327, partially reverses TGF-β-induced EMT in an immortalized mouse mammary epithelial cell line. Herein, we demonstrate that ML327 reverses much of the EMT gene expression program in cultured carcinoma cell lines. The reversal of EMT sensitizes these cancer cells to the apoptosis-inducing ligand TRAIL. This sensitization is independent of E-cadherin expression and rather relies on the downregulation of a major anti-apoptotic protein, cFLIPS. Loss of cFLIPS is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIPS restores resistance to TRAIL-induced apoptosis despite EMT reversal with ML327. In summary, we have utilized an isoxazole-based small molecule that partially reverses EMT in carcinoma cells to demonstrate that cFLIPS critically regulates the apoptosis resistance phenotype associated with EMT.

Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation.

Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine acetylation profiles for particular biological states, but none to date have investigated the temporal dynamics regulating cellular response to perturbation. Reasoning that lysine acetylation may be altered in response to growth factors, we implemented quantitative mass spectrometry-based proteomics to investigate the temporal dynamics of lysine acetylation in response to growth factor stimulation in cultured carcinoma cell lines. We found that lysine acetylation changed rapidly in response to activation of several different receptor tyrosine kinases by their respective ligands. To uncover the effects of lysine acetylation dynamics on tyrosine phosphorylation signaling networks, cells were treated with an HDAC inhibitor. This short-term pharmacological inhibition of histone deacetylase activity modulated signaling networks involving phosphorylated tyrosine and thereby altered the response to receptor tyrosine kinase activation. This result highlights the interconnectivity of lysine acetylation and tyrosine phosphorylation signaling networks and suggests that HDAC inhibition may influence cellular responses by affecting both types of post-translational modifications.

Dickkopf‐1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK-1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK-1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK-3 expression was also seen. In contrast, the expression of DKK-2 and -4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK-1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK-1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK-1 in pancreatic carcinoma cells, we performed a knockdown of DKK-1 in SUIT-2 human pancreatic adenocarcinoma cell line and S2-CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK-1 knockdown resulted in reduced migratory activity of SUIT-2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK-1 knockdown in S2-CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK-1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

Metabolomic studies of human lung carcinoma cell lines using in vitro1H NMR of whole cells and cellular extracts

We report principal component analysis (PCA) of (1)H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and (1)H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid (1)H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the (1)H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results.

Refractory Factors in Head and Neck Cancer: ATP Binding Cassette Transporters Expressed in Head and Neck Cancer Cell Lines

The aim of the present study was to clarify whether ATP binding cassette transporters are refractory factors in head and neck cancer chemotherapy. For in vitro and in vivo chemotherapeutic studies, we employed a human salivary gland adenocarcinoma cell line (HSY) and a human oral squamous cell carcinoma cell line (SCCSK) with vincristine (VCR) at clinically equivalent doses. Western blot analysis, reverse transcription-polymerase chain reaction, in vivo evaluation in xenograft models inoculated with cultured carcinoma cell line and drug efflux analysis were performed. VCR-treated SCCSK and HSY cells, as well as xenografted SCCSK and HSY cells in tumor-bearing nude mice, were found to express MDR1/ABCB1 and MRP1/ABCC1. In addition to MDR1 and MRP1 mRNA, HSY/VCR and its cloned cells expressed MRP7/ABCC10 mRNA, but SCCSK/VCR did not express MRP7. Furthermore, drug resistance to VCR and docetaxel decreased in HSY/VCR in the presence of a competitive MRP7 inhibitor, 17-beta-estradiol-(17-beta-D-glucuronide). These results indicate that MDR1 and MRP1 expression are refractory factors in head and neck cancer chemotherapy and suggest that induction of MRP7 expression is involved in drug resistance in salivary gland adenocarcinomas.

Cytochrome P450 2J2 Promotes the Neoplastic Phenotype of Carcinoma Cells and Is Up-regulated in Human Tumors

Cytochrome P450 (CYP) arachidonic acid epoxygenase 2J2 converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids, which exert diverse biological activities in cardiovascular system and endothelial cells. However, it is unknown whether this enzyme highly expresses and plays any role in cancer. In this study, we found that very strong and selective CYP2J2 expression was detected in human carcinoma tissues in 101 of 130 patients (77%) as well as eight human carcinoma cell lines but undetectable in adjacent normal tissues and nontumoric human cell lines by Western, reverse transcription-PCR, and immunohistochemical staining. In addition, forced overexpression of CYP2J2, and CYP BM3F87V or addition of epoxyeicosatrienoic acids (EET) in cultured carcinoma cell lines in vitro markedly accelerated proliferation by analyses of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell accounts, and cell cycle analysis, and protected carcinoma cells from apoptosis induced by tumor necrosis factor alpha (TNF-alpha) in cultures. In contrast, antisense 2J2 transfection or addition of epoxygenase inhibitors 17-ODYA inhibited proliferation and accelerated cell apoptosis induced by TNF-alpha. Examination of signaling pathways on the effects of CYP2J2 and EETs revealed activation of mitogen-activated protein kinases and PI3 kinase-AKT systems and elevation of epithelial growth factor receptor phosphorylation level. These results strongly suggest that CYP epoxygenase 2J2 plays a previously unknown role in promotion of the neoplastic cellular phenotype and in the pathogenesis of a variety of human cancers.

Histone deacetylase inhibitors upregulate plakoglobin expression in bladder carcinoma cells and display antineoplastic activity in vitro and in vivo

Histone deacetylase inhibitors (HDACis) are emerging as a promising new class of anticancer agents displaying growth-inhibitory activity and low toxicity in vivo. In this study, we examined the effect of sodium butyrate (NaB) and trichostatin A (TSA) on the growth of human bladder carcinoma cell lines in culture and TSA on the growth of EJ and UM-UC-3 human bladder xenografts in nude mice. NaB and TSA suppressed the growth of bladder cell lines at millimolar (1.5-4.3 mM) and micromolar (0.03-0.33 microM) concentrations, respectively, inducing concentration-dependent cell death. Bladder carcinoma cells within the experimental panel displayed the phenotype of late-stage bladder lesions expressing N-cadherin in the absence of E-cadherin accompanied by low levels of plakoglobin expression. Exposure of these cells to HDACis resulted in upregulation of plakoglobin with no change in E-cadherin expression. A 2-hr exposure to TSA was the minimal time required to upregulate plakoglobin in cells with downregulation to baseline levels occurring within 24 hr following drug removal. In mice bearing EJ and UM-UC-3 bladder xenografts, TSA (500 microg/kg/day) caused suppression of tumor growth compared with mice receiving vehicle alone. A > 70% reduction in mean final tumor volume was recorded in both bladder xenograft models with no detectable toxicity. The results suggest that TSA inhibits bladder carcinoma cell growth and may be a useful, relatively nontoxic agent for consideration in the treatment of late-stage bladder tumors.

296. Combination Therapy with Adenovirus (Ad5) and Cytotoxic Drugs Can Act Synergistically To Induce Cell Death in Carcinoma Cell Lines in Culture

Current breast cancer (BC) treatments such as chemotherapy and radiotherapy has low specificity for cancer cells, thus also damage normal tissues. Cancer-targeting gene therapy has showed great promise to minimize treatment side effects and the risk of developing resistance. To develop a specific promoter for BC-targeting gene therapy, we identified six candidate BC-specific genes from published gene expression profile data. The promoter of one of them was shown to possess BC-specific activity in vitro, and named as BCSP. Deletion mutant analysis showed that the minimal promoter of BCSP retained BC-specific activity. In order to increase promoter activity for clinical use in gene therapy, an enhancer sequence from a promoter known to have strong activity was connected to the minimal BCSP, and the construct was named enhanced BC-specific promoter, EBP. To determine the activity of EBP, we performed reporter assays in cell lines and an orthotopic BC xenograft mouse model. In mouse model, EBP activity was higher than that of CMV promoter in tumor, and less than 10% of CMV promoter activity in normal organs such as lung and heart. Similar results were seen in cell lines. These results suggested that EBP is a strong BC-specific promoter for gene therapy. To test its therapeutic efficacy, EBP was used to drive the expression of BikDD (Bik mutant with more potent pro-apoptotic activity). We found that this therapeutic construct, EBP-BikDD, selectively killed BC cells rather than normal breast epithelial cells, in in vitro viability assay. The therapeutic effect of the liposome-complexed EBP-BikDD was then evaluated in orthotopic BC xenograft mouse model through intravenously injection. EBP-BikDD suppressed tumor growth and extended the life span of tumor-bearing mice significantly. Moreover, in situ hybridization showed that both EBP and the CMV promoter drove mRNA expression of BikDD in breast tumors, but EBP induced much lower BikDD expression in normal organs (e.g. heart) than CMV did. In conclusion, we have developed a strong and highly specific promoter for BC-targeting gene therapy. This therapy effectively suppresses human breast tumor growth in animal model and is much less toxic for normal tissues than the CMV promoter, which is widely used in current cancer gene therapy.

Comparison of differentiation markers between normal and two squamous cell carcinoma cell lines in culture

This study examines differences between cultures of normal human oral epithelial cells and two squamous cell carcinoma cell lines (SCC15 and SCC25) in the expression of structural proteins, adhesion molecules, plasma membrane lipid composition, and intercellular junctions. Based on immunocytochemistry, most normal cell cultures appeared to express more E-cadherin, integrin β-1, cytokeratin (CK) 14, CK19, and involucrin than SCC cultures. By Western blot analysis, normal cultures expressing high levels of E-cadherin also expressed high levels of involucrin and low levels of CK19. Both SCC cultures demonstrated lower expression of E-cadherin and involucrin, whereas only SCC15 cells showed high levels of CK19. Expression of β-catenin, an E-cadherin associated protein with potential oncogene function, did not vary among normal and SCC cells. Proportions of saturated fatty acids quantified by thin layer chromatography were higher in the normal cell cultures, than in both SCC cell lines. No morphological differences were evident by transmission electron microscopy (TEM) between normal and SCC cell–cell intercellular junctions. Although no quantitation was attempted, observation suggested that normal cells form more intercellular junctions (TEM observation) and larger intercellular bridges (SEM observation) compared to both SCC cell lines. Of the factors examined, main variations between cultures of normal oral epithelium and the two SCC cell lines examined include the expression of structural and adhesion proteins, lipid composition, and intercellular junctions. The extent of the differences varies according to the stage of terminal differentiation demonstrated by the normal cell cultures.

The nuclear oxysterol receptor LXRalpha is expressed in the normal human breast and in breast cancer.

The liver X> or = receptor alpha (LXRalpha) is a nuclear receptor with a key role in bile acid biosynthesis and cholesterol metabolism. The present study investigated the expression and function of LXRalpha in the normal and malignant human breast. LXRalpha mRNA transcripts were detected by RT-PCR in nine breast carcinoma cell lines. The nucleotide sequence of the cloned PCR product was identical to the corresponding human LXRalpha cDNA sequence. Expression of LXRalpha protein was confirmed by immunoblot analysis of breast cancer cell lysates. LXRalpha mRNA was expressed in 14/15 (93%) of normal human breast mammoplasty specimens and in 11/15 (73%) of primary breast carcinomas. Oxysterol and nonsteroidal LXRalpha agonists at low micromolar concentrations inhibited proliferation of breast carcinoma cell lines in culture. The importance of LXRalpha signaling in cholesterol homeostasis and the observed expression of LXRalpha in normal breast tissue suggest that this nuclear oxysterol receptor has an important physiological function in the breast. LXRalpha gene expression is regulated by dietary fatty acids implicated in breast carcinogenesis and detection of LXRalpha expression in breast cancer cell lines and breast tumors in the present study indicates that LXRalpha may also be important in breast carcinogenesis. Inhibition of breast cancer cell proliferation suggests that pharmacological LXRalpha agonists may have potential preventive and/or therapeutic antitumor activity in breast cancer.

Freshly isolated bone marrow cells induce death of various carcinoma cell lines.

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival.

Angiogenic factors produced by tumor cells are essential for tumor growth and metastasis. In our study, the expression of Angiopoietin-1 (ANG1) and Angiopoietin-2 (ANG2) mRNA in archival human breast cancer tumor samples and in 6 breast cancer cell lines was investigated. Total RNA from biopsies of 38 breast cancer patients was extracted and ANG1 and ANG2 mRNA expression was measured by means of quantitative real-time RT-PCR (Taqman). Matching data with available clinicopathologic and biochemical data revealed a significant association between ANG2 expression and axillary lymph node invasion. Univariate and multivariate survival analysis, by means of Kaplan-Meier method and Cox's proportional hazards model, showed significant and independent association between ANG2 mRNA level and both disease-free (p < 0.0001) and overall survival (p < 0.0003). An important fact is that, notwithstanding the small number of cases examined, this association was confirmed also in the group of lymph node-negative patients (DFS, p < 0.003; OS, p < 0.020). Immunohistochemical analysis demonstrated that Ang2 is expressed by both tumor cells and endothelial elements. Expression in tumor cells was confirmed by studying a panel of human breast carcinoma cell lines in culture by RT-PCR. In ZR75.1 and T47D cells, expression of ANG2 mRNA was increased up to 10-fold by treatment with estrogen within 24 hr. Although preliminary, these data suggest a possible role of ANG2 as a prognostic factor for primary breast cancer.

Role of Peroxisome Proliferator-Activated Receptor γ and Its Ligands in Non-Neoplastic and Neoplastic Human Urothelial Cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is expressed in several types of tissue. Although PPARgamma reportedly is expressed in normal urothelium, its function is unknown. We examined the expression of PPARgamma in normal urothelium and bladder cancer in an attempt to assess its functional role. Immunohistochemical staining revealed normal urothelium to express PPARgamma uniformly. All low-grade carcinomas were positive either diffusely or focally, whereas staining was primarily focal or absent in high-grade carcinomas. A nonneoplastic urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell line, and two high-grade (T24 and 253J) carcinoma cell lines in culture expressed PPARgamma mRNA and protein. Luciferase assay indicated that PPARgamma was functional. PPARgamma ligands (15-deoxy-Delta(12,14)-prostaglandin J(2), troglitazone and pioglitazone) suppressed the growth of nonneoplastic and neoplastic urothelial cells in a dose-dependent manner. However, neoplastic cells were more resistant than were nonneoplastic cells. Failure to express PPARgamma or ineffective transcriptional activity may be some of the mechanisms responsible for resistance to the inhibitory action of PPARgamma ligands.

αvβ3 and Vitronectin Expression by Normal Ovarian Surface Epithelial Cells: Role in Cell Adhesion and Cell Proliferation

Theαvβ3 integrin and its ligand vitronectin are expressed by differentiated epithelial ovarian carcinomas and carcinoma cell lines in culture. Moreover, αvβ3/vitronectin interaction influences adhesion and migration of ovarian carcinoma cells in culture. For a better understanding of the behavior of these carcinomas, it appeared necessary to study the characteristics of their normal counterpart, the ovarian surface epithelium (OSE). The present study showed that normal cultured human OSE cells, like the carcinoma cells, have the ability to synthesize vitronectin. The vitronectin receptor, αvβ3 integrin, is also expressed by OSE cells and is localized in focal contacts close to paxillin, a focal contact-specific protein, and p125FAK, a cytoskeletal and signaling molecule. This localization suggested an active participation of the integrin in the adhesion and/or proliferation of OSE cells. Indeed, the use of a blocking antibody demonstrated that αv integrins promote OSE cell adhesion on vitronectin but not on fibronectin and that these integrins are required for maximal proliferative activity. The results suggest a role of the αvβ3/vitronectin system in normal OSE physiology and demonstrate that the expression of this system by well-differentiated ovarian carcinomas reflects the retention of normal cell properties.

Galectin-7, a marker of all types of stratified epithelia

Galectin-7 is a 14-kDA member of the lectin family we have previously cloned in the human. Its expression was found at all stages of differentiation of the human epidermis and was reduced but not suppressed when oral metaplasia of reconstructed epidermis was induced by retinoic acid. This suggested that galectin-7 could be a marker of both keratinized and non-keratinized stratified epithelia. To ascertain this hypothesis, we cloned the rat and the mouse cDNAs and produced a specific antiserum raised against a synthetic peptide. The distribution of galectin-7 mRNAs and protein was studied by in situ hybridization and immunolabelling of various human, rat and mouse epithelia. Galectin-7 was found to be expressed in interfollicular epidermis and in the outer root sheath of the hair follicle, but not in the hair matrix, nor in the sebaceous glands. It was present in esophagus and oral epithelia, cornea, Hassal's corpuscles of the thymus, but not in simple and transitional epithelia. Galectin-7 can thus be considered as a marker of all subtypes of keratinocytes. In that respect it differs from both “basal-specific” keratins K5–K14 and from “suprabasal-specific” markers such as keratins K1–K10 and involucrin. Galectin-7 also differs from some desmosomal proteins, which are present in all types of epithelia and in myocardium. Galectin-7 was absent from cultured carcinoma cell lines and was reduced both in human carcinomas and in murine tumors produced with the two-stage carcinogenesis protocol.

Inhibition of proliferation and induction of apoptosis in cervical carcinoma cells by retinoids: Implications for chemoprevention

The effects of retinoids including all-trans-retinoic acid (ATRA), 13-CIS-RETINOIC ACID (13CRA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on several cervical carcinoma cell lines in culture were investigated as a prelude to investigating the mechanisms underlying the chemopreventive potential of retinoids in cervical cancer. We found that when used at a concentration of 1 microM, 13CRA and ATRA inhibited the proliferation of three cell lines (ME-180 [HPV 68], SiHa [HPV 18], and HT-3 [HPV-]) by about 80% after a seven-day treatment. Three other cell lines (MS-751 [HPV 18], HeLa [HPV 18], C-33A [HPV-]) were moderately inhibited (30-48%), and two (C-4 II [HPV 18], CaSki [HPV 16]) responded poorly (< 25% inhibition). 4-HPR failed to inhibit the growth of any of these cell lines when used at 1 microM; however, when used at 5 or 10 microM, it induced apoptosis as evidenced by DNA fragmentation in several of the cell lines and was more potent in this effect than 10 microM ATRA. Retinoids that induce apoptosis in malignant cells may be able to exert similar effects on premalignant cells. Such retinoids would be expected to exhibit greater potency as chemopreventive agents than retinoids that exert only cytostatic effects.

Effect of radiation combined withhyperthermia on human prostatic carcinoma cell lines in culture

The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses followed by heat treatment at 43 degrees C for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation and heat produced greater cytotoxicity than predicted from the additive effects of the two individual treatment modalities alone. These results indicate that a combined treatment regimen of radiation plus hyperthermia (43 degrees, 1 hr) might be an important tool in maintaining a better local control of prostatic cancer.

Alterations in c-myc expression in relation to maturational status of human colon carcinoma cells.

Previous work from this laboratory established a large bank of human colon carcinoma cell lines in culture and classified them with respect to growth regulatory phenotypes based on several biological and biochemical characteristics. In the present report, Northern analysis indicates that addition of the maturational agent N,N-dimethylformamide (DMF) (1.0%) to proliferating HCT 116 and MOSER cells resulted in a repression of c-myc proto-oncogene expression; retinoic acid (1.0 microM) was less effective in this regard. Repression of c-myc expression by DMF was observed in MOSER and HCT 116 cells, whether it was added to proliferating or late log phase cultures, and was associated with a corresponding reduction in cellular proliferation. The reduction in c-myc expression by DMF did not require new protein synthesis and occurred within a few minutes after its addition, resulting in a 70% reduction within approximately 2 hr. Previous work from this laboratory indicated that transforming growth factor-beta (TGF-beta) elicited alterations in MOSER cells similar to those observed following DMF treatment. The present report demonstrates that proliferating, but not late log phase, MOSER cells responded to TGF-beta with a repression of c-myc expression. Similarly, an inhibition of cellular proliferation was only observed when TGF-beta (10 ng/ml) was added to proliferating cells. Collectively, these findings indicate that repression of c-myc expression is associated with diminished cellular proliferation and the induction of a more benign phenotype in human colon carcinoma cells. Furthermore, this report is the first demonstration of a c-myc response to TGF-beta in an epithelial cell line.

Vasoactive intestinal peptide: A potent stimulator of adenosine 3′:5′-cyclic monophosphate accumulation in gut carcinoma cell lines in culture

Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3x10(-12) M. Maximum VIP-induced cAMP levels were observed with 10(-9) M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3x10(-10) M VIP. (125)I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10(-10) and 10(-7) M. Half-maximum inhibition of binding was observed with 2x10(-9) M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10(-7) M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E(1) and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10(-8) M; half-maximum stimulation was observed at about 10(-9) M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation.