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At least 433 records · Page 24Linked to original sources
PubMed1999

Altered phenotype of HT29 colonic adenocarcinoma cells following expression of the DCC gene.

On 18q, frequently deleted in late stage colorectal cancers, a gene, Deleted in Colon Cancer (DCC), has been identified and postulated to play a role as a tumor suppressor gene. DCC is retained in the majority of mucinous tumors, which produce high levels of mucins, and seems to be preferentially expressed in intestinal goblet cells. To investigate whether DCC is related to mucin expression and can modulate the transformed phenotype, we introduced a full-length DCC cDNA into HT29 cells, which can be induced in vitro to express MUC2, the gene that encodes the major colonic mucin. Expression of DCC did not modulate constitutive or induced expression of MUC2, nor did DCC induce a mature goblet cell phenotype. However, HT29 clones expressing high and low levels of DCC protein showed a significant decrease in cell proliferation and tumorigenicity. Furthermore, increased shedding and an elevated rate of spontaneous apoptosis were associated with higher levels of expression of DCC. In summary, while restoration of DCC expression in a human colon carcinoma cell line did not influence expression of differentiation markers, DCC expression did affect the growth and tumorigenic properties of the cells suggesting that DCC can modulate the malignant phenotype of colon cancer.

Adenocarcinoma↗
PubMed2004

Tetrandrine induces early G1 arrest in human colon carcinoma cells by down-regulating the activity and inducing the degradation of G1-S-specific cyclin-dependent kinases and by inducing p53 and p21Cip1.

Tetrandrine is an antitumor alkaloid isolated from the root of Stephania tetrandra. We find that micromolar concentrations of tetrandrine irreversibly inhibit the proliferation of human colon carcinoma cells in MTT and clonogenic assays by arresting cells in G(1). Tetrandrine induces G(1) arrest before the restriction point in nocodazole- and serum-starved synchronized HT29 cells, without affecting the G(1)-S transition in aphidicolin-synchronized cells. Tetrandrine-induced G(1) arrest is followed by apoptosis as shown by fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated nick end labeling, and annexin V staining assays. Tetrandrine-induced early G(1) arrest is mediated by at least three different mechanisms. First, tetrandrine inhibits purified cyclin-dependent kinase 2 (CDK2)/cyclin E and CDK4 without affecting significantly CDK2/cyclin A, CDK1/cyclin B, and CDK6. Second, tetrandrine induces the proteasome-dependent degradation of CDK4, CDK6, cyclin D1, and E2F1. Third, tetrandrine increases the expression of p53 and p21(Cip1) in wild-type p53 HCT116 cells. Collectively, these results show that tetrandrine arrests cells in G(1) by convergent mechanisms, including down-regulation of E2F1 and up-regulation of p53/p21(Cip1).

Alkaloids↗
PubMed1988

Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line.

HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this report to be a model system for the study of human goblet cell differentiation and mucin secretion. Grown in the absence of glucose, these cells formed homogeneous epithelial monolayers of columnar cells with typical goblet cell morphology. Differentiation occurred on uncoated glass; laminin, fibronectin, or collagen type I or IV did not enhance differentiation. HT29-18N2 cells grown on uncoated or matrix-coated permeable filters formed differentiated monolayers, but mucin granules within some of these cells polarized along intraepithelial lumens. Polyclonal antibodies raised against purified human colonic mucin, and also a monoclonal antibody against a protease-sensitive epitope of human colonic mucin, stained secretory granules of all differentiated goblet cells within N2 cell monolayers but did not stain predifferentiated goblet cells lacking large secretory granules. Monoclonal antibodies against specific carbohydrate sequences of human mucins also failed to stain N2 cells before differentiation, but recognized varying fractions of differentiated N2 goblet cells. Autoradiographic visualization of radiolabeled glycoproteins demonstrated transport and secretion of N2 cell mucin granules. Cholinergic stimulation of differentiated N2 cell monolayers resulted in depletion of intracellular mucin granules.

Adenocarcinoma↗
PubMed2005

SKI-606, a Src/Abl inhibitor with in vivo activity in colon tumor xenograft models.

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.

Administration, Oral↗
PubMed1998

Synthesis and cytotoxic activity of pyranophenanthridine analogues of fagaronine and acronycine.

Condensation of 5-amino, 6-amino, 7-amino and 8-amino-2,2-dimethyl-2H-chromenes with either 6-bromoveratraldehyde or 6-chloropiperonal afforded the corresponding Schiff bases, which were subsequently reduced to the corresponding benzylchromenylamines 30-33 and 36-39. Lithium diisopropylamide-mediated cyclization of those amines, followed by spontaneous air oxidation, afforded pyranophenanthridines 3-14. The cytotoxicity of compounds 3-14 was evaluated against L1210 and HT29 cell lines. 9,9-Dimethyl-9H-pyrano[3,2-b]phenanthridines appear to be the most promising compounds of the series, since both the dimethoxy derivative 11 and the methylenedioxy derivative 12 exhibit significant cytotoxic activity. Compound 12 was the most active and induced a massive accumulation of cells in G2 + M phases, suggesting that the cytotoxicity is due to a perturbation of the integrity or function of DNA.

Acronine↗
PubMed1991

Differences in lipid characteristics of undifferentiated and enterocytic-differentiated HT29 human colonic cells.

In this paper we compared several lipid characteristics of the homogenate and the corresponding plasma membrane in undifferentiated and differentiated HT29 human colon cancer cells, using normal human colonic cells as a reference. Electron microscopy showed that HT29 cells were morphologically undifferentiated when cultured in the presence of either glucose or inosine without glucose at early confluency. On the contrary, HT29 cells cultured at late confluency in a glucose-free medium containing inosine or grown in nude mice exhibited an enterocytic differentiation with the presence of tight junctions and an apical brush border. The cell homogenate and the plasma membrane were prepared from each cell type. The study of specific marker enzymes showed the same degree of purity in all plasma membranes, with a highly marked increase of brush border-associated hydrolases (N-aminopeptidase and alkaline phosphatase) only in the organelles isolated from differentiated HT29 and colonic cells. Respective similar increases in the amount of free cholesterol and phospholipid and in the free cholesterol:phospholipid molar ratio were found in the plasma membrane as compared with the homogenate in all HT29 cell types. This ratio, due to an increased phospholipid content in both homogenate and plasma membrane, was lowered in colonic cells. No differences in the phospholipid profile were found between the homogenates of all cell types and the plasma membrane of undifferentiated HT29 cells, with the exception of a decrease of cardiolipin in this organelle. On the contrary, the plasma membrane phospholipid composition was different from that of the corresponding homogenate in differentiated HT29 and colonic cells. The most striking changes were a highly increased sphingomyelin amount and concomitant decreases in phosphatidylethanolamine, phosphatidylserine, and cardiolipin. Moreover, differences in the percentage of phosphatidylcholine plus sphingomyelin as well as in phosphatidylcholine:sphingomyelin, phosphatidylethanolamine, and/or phosphatidylcholine molar ratios were also found. The monounsaturated:polyunsaturated fatty acid ratio in phosphatidylethanolamine was similar in differentiated HT29 and colonic cells and lower than in undifferentiated HT29 cells. A decrease in this latter ratio in phosphatidylcholine was also observed in colonic cells and HT29 cells grown in nude mice. These changes were essentially due to opposite variations in the percentage of palmitoleic acid and those of linoleic and/or arachidonic acids in both phospholipids. Thus, these data indicate that undifferentiated HT29 cells were characterized by the absence of a specific phospholipid composition in their plasma membrane, which is suggested to be related to altered phospholipid sorting. The plasma membrane phospholipid profile reversed essentially to the normal pattern when HT29 cells recovered the ability to differentiate.

5'-Nucleotidase↗
PubMed1996

Alpha 2-adrenergic agonists increase cellular lactate efflux.

We reported previously that genetic polymorphisms of the alpha 2-adrenergic receptor are associated with hyperinsulinemia, diabetes mellitus, and hypertension in blacks. The evolutionary driving force for maintaining such deleterious mutations in the black population is unknown. Recognizing that vascular alpha 2-adrenergic receptors mediate cold-induced vasoconstriction and that temperature maintenance is a primary thrust of cellular metabolism, we postulated that vascular alpha 2-adrenergic receptors contribute significantly to metabolic heat generation in homeotherms such as humans. Using aerobic lactate production as an indicator of thermogenesis, we measured metabolic heat production in HT29 cells that expressed the gene encoding human vascular alpha 2-adrenergic receptors. Epinephrine, an alpha 2-adrenergic receptor agonist, increased net lactate efflux from 226 +/- 20 to 280 +/- 20 nmol/min (mean +/- SE) (P = .06). Clonidine, a more specific alpha 2-adrenergic agonist, increased lactate efflux from 110 +/- 6 to 156 +/- 8 nmol/min (P < .01). Similarly, in the presence of physiological concentrations of glucose (5.5 mmol/L), insulin increased lactate production from 123 +/- 6 to 175 +/- 10 nmol/min (P < .01). Because differences in aerobic glycolysis may also explain the heat intolerance and abnormal fuel homeostasis found in genetically hypertensive rats, we also measured lactate production in cultured vascular smooth muscle cells isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control Wistar-Kyoto rats (WKY). Vascular smooth muscle cells from SHRSP had significantly greater lactate efflux compared with cells from normotensive WKY (296 +/- 4 versus 172 +/- 2 nmol/min, P < .001). These differences were not due to abnormalities in glucose uptake, as lactate efflux was greater in SHRSP cells compared with WKY cells when dextrose was replaced with equimolar concentrations of fructose (230 +/- 6 versus 138 +/- 2 nmol/min, P < .001). alpha 2-Adrenergic agonists increase lactate efflux in HT29 cells, and abnormalities in vascular smooth muscle lactate metabolism in genetically hypertensive rats is independent of altered glucose uptake. These data provide support for our hypothesis that balanced polymorphisms of the alpha 2-adrenergic receptor could offer protection against cold stress by increasing the thermogenic response associated with aerobic lactate production.

Adrenergic alpha-Agonists↗
PubMed1999

Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.

Apoptosis↗
PubMed2004

P21Cip1 is a critical mediator of the cytotoxic action of thymidylate synthase inhibitors in colorectal carcinoma cells.

We have demonstrated previously that interferon (IFN)-gamma sensitizes human colon carcinoma cell lines to the cytotoxic effects of 5-fluorouracil combined with leucovorin and to the thymidylate synthase inhibitor, ZD9331, dependent on thymineless stress-induced DNA damage, independent of p53. Here we demonstrate that the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) regulates thymineless stress-induced cytotoxicity in these cells. HCT116 wild-type (wt) and p53-/- cells underwent apoptosis and loss in clonogenic survival when exposed to ZD9331, whereas p21Cip1-/- cells were resistant. In contrast, IFN-gamma induced marked cytotoxicity in p21Cip1-/- cells only. ZD9331 induced p21Cip1 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymidylate synthase-deficient (thymidylate synthase-) cells. Furthermore, selective induction of p21Cip1 in RKO was sufficient to induce apoptosis. P21Cip1, cdk1, cdk2, and cyclin E mRNA expression increased coincident with S-phase accumulation in HT29 cells treated with ZD9331 or 5fluorouracil/leucovorin, as demonstrated by cDNA microarray analyses. Cell cycle analyses revealed that HCT116 wt and p21Cip1 -/- cells accumulated in S phase within 24 h of ZD9331 exposure; however, wt cells exited S-phase more rapidly, where apoptosis occurred before mitosis, either in late S or G2. Finally, the CDK inhibitor roscovitine potentiated the cytotoxic activity of ZD9331 in both wt and p21Cip1-/- cells, strongly suggesting a role for p21Cip1-dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21Cip1 positively regulates the cytotoxic action of thymidylate synthase inhibitors, negatively regulates the cytotoxic action of IFN-gamma, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.

Apoptosis↗
PubMed2001

[Inhibition of the growth of hepatoma and hepatic metastasis by pingyangmycin conjugated with Fab' fragment of monoclonal antibody].

OBJECTIVE: To develop an immunoconjugate with inhibitory effect on the growth of hepatoma and hepatic metastasis of tumor by linking Fab' fragment of monoclona antibody (McAb) to pingyangmycin (PYM). METHODS: Monoclonal antibody Fab' fragment was obtained by enzymolysis with pepsin and DTT reduction. Linking of the Fab' fragment and PYM was mediated by dextran T40. Indirect ELISA, TTC bacteriostatic titer test, clonogenic assay and animal models transplanted with tumor were used to assess the biological activities and antitumor effects of Fab' PYM conjugate within and without the bodies of 10 mice. RESULTS: The Fab' PYM conjugate showed immunoreactivity to BEL7402, hepatoma 22 (H22) and HT29 cells, but no reaction to KB cells. The bacteriostatic activity of PYM in the conjugate was 15% as much as that of free PYM. The 50% inhibitory doses (IC50) of Fab'PYM conjugate to BEL7402, HT29 cells and KB cells were 0.02 micromol/L, 0.08 micromol/L and 0.50 micromol/L respectively. Given intravenously, 10 mg/kg x 6, Fab' PYM conjugate and PYM suppressed the growth of hepatoma 22 by 86% and 62% respectively. Given intraperitoneally, 10 mg/kg x 4, Fab' PYM and PYM decreased the hepatic metastasis of colon carcinoma 26 by 91% and 62% respectively. CONCLUSION: Fab' PYM conjugate is more effective in combating hepatoma and hepatic metastasis of tumor than free PYM.

Animals↗
PubMed2004

Limited tumor growth (HT29) in vivo under RO205-2349 is due to increased apoptosis and reduced cell volume but not to decreased proliferation rate.

The peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that plays a regulatory role in cell differentiation and proliferation. PPARgamma was first detected in adipocytes, however, it has been shown that this receptor is also expressed in normal as well as tumor cells including malignant colonic epithelial cells. In this study, the effect of the PPARgamma agonist RO205-2349, a recently developed thiazolidinedione, on tumor growth was evaluated. For this purpose, human colon cancer cells (HT29) were grown in severe combined immunodeficient mice. Under daily RO205-2349 treatment (50 mg/kg/day) a significantly reduced tumor weight became evident after 3 weeks. In the control (n = 10) and treatment (n = 10) groups the mean tumor weights were 0.45 and 0.16 g, respectively. The mean percentages of apoptotic cells were 0.8 and 2.7% in the control and treatment groups, respectively, and the cell diameter measured on average 11.4 and 9.4 microm. In contrast, cell proliferation and differentiation, which are considered to be influenced by the PPARgamma, remained unaffected as could be seen by Ki-67 and carcinoembryonic antigen immunoreactivity indicating that increased rate of apoptosis and cell shrinkage are responsible for the differences in tumor growth. Hence, in this human/mouse xenograft model, mechanisms other than the classical activation of PPARgamma are likely reasons causing limited tumor growth.

Animals↗
PubMed2000

Inhibitory effect of ATF3 antisense oligonucleotide on ectopic growth of HT29 human colon cancer cells.

ATF3 is a transcription factor belonging to the Jun / Fos family whose mouse homologue (TI-241) was isolated, using the differential screening method, from B16 mouse melanoma cells as a blood-borne metastasis-associated gene. Here we show the tumorigenicity-inhibiting effect of an antisense oligonucleotide designed to reduce the expression of ATF3 in HT29 colon cancer cells. HT29 cells were reported to metastasize to the skin after intravenous inoculation. The antisense oligonucleotide inhibited cell attachment to the collagen-coated floor of the plates and invasion of HT29 cells in vitro, which are thought to be two important factors in the process of cancer metastasis and ectopic tumor growth. While the antisense oligonucleotide had no effect on cell growth of HT29 cells in vitro, mice that were inoculated subcutaneously with HT29 cells and treated with the antisense oligonucleotide survived longer than the control mice due to the inhibition of tumor growth in vivo. These show that ATF3 plays an important role in the ectopic growth / metastasis of HT29 colon cancer cells.

Activating Transcription Factor 3↗
PubMed1997

The 3' untranslated region of the carcinoembryonic antigen gene plays a minimal role in the regulation of gene expression.

Carcinoembryonic antigen (CEA), an oncofetal glycoprotein, is overexpressed in both colonic tumor tissue and in aberrant crypt foci. The 3' untranslated region (UTR) of CEA, which has been postulated to play a role in CEA expression, contains an oligo-A region. Sequence instability at oligo-A regions occurs in colorectal cancer, and it has been suggested that these mutations may alter expression of genes involved in the development of colonic tumors. We demonstrate that the oligo-A region of the CEA 3' UTR does in fact exhibit sequence instability. The significance of this instability, and of this region of the CEA 3' UTR in general, to CEA expression was analyzed in the HT29 cell line, colonic tumor cells which overexpress CEA upon induction with sodium butyrate. CEA promoter constructs, containing sense or antisense versions of the normal or mutant CEA 3' UTR region, were transfected into uninduced and induced HT29 cells. Although we could demonstrate that activity of the exogenous CEA promoter was markedly elevated in cells induced to differentiate, the data demonstrate no sequence or orientation-specific effect of the 3' UTR of CEA on expression from a CEA promoter. In addition, instability of the 3' UTR of CEA could be demonstrated in LoVo cells. However, there is no marked over- or underexpression of mutated 3' UTR CEA sequences in this cell line. We conclude that the oligo-A-containing region of the 3' UTR of CEA (204 bp beginning at nucleotide 24229) does not play a substantial role in the regulation of CEA expression.

Aged↗
PubMed2000

Purinergic responses in HT29 colonic epithelial cells are mediated by G protein alpha -subunits.

Using Fura-2 to measure changes in intracellular calcium ([Ca(2+)](i)), we show that P(2U)receptors in HT29 cells trigger an increase in [Ca(2+)](i)by pertussis toxin-insensitive G proteins. We then use replication-deficient adenoviruses expressing wild-type and dominant negative mutants of G(alpha q)and G(alpha i2), antisense directed against G(alpha q)or the C-terminal fragment of beta-adrenergic receptor kinase (beta ARK-CT) to identify these G proteins. We find the [Ca(2+)](i)response to UTP is not affected by increased expression of the wild-type G(alpha q), wild-type G(alpha i2)or beta ARK-CT, while it is blocked by over-expression of dominant negative G(alpha q). The timecourse of the UTP response is, however, altered by wild-type G(alpha q)and is only weakly inhibited by antisense G(alpha q). This suggests that the P(2U)response is mediated, at least partially, by a G protein distinct from G(alpha q). In contrast, the M(3)muscarinic response is inhibited by over-expression of antisense against G(alpha q), or over-expression of beta ARK-CT, a finding in agreement with our previous observation that the muscarinic response in HT29 cells is mediated by the beta gamma-subunits of G(q). We also find that P(2U)and M(3)receptors do not control identical Ca(2+)stores, suggesting that differential activation of G proteins can lead to Ca(2+)release from distinct stores.

Adenoviridae↗
PubMed1998

Modulation of hepatocyte growth factor-induced scattering of HT29 colon carcinoma cells. Involvement of the MAPK pathway.

Hepatocyte growth factor (HGF)/scatter factor modulates the motility of HT29 colon carcinoma cells in vitro by inducing morphological changes that depend on the type of extra-cellular matrix (ECM) ligand; HGF-induced scattering of HT29 cells is observed if cells are grown on plastic coated with serum proteins but not laminin. The absence of scattering correlates with a lack of cell spreading on laminin and it is not due to impaired HGF induced tyrosine phosphorylation of the E-cadherin/desmosome component, (gamma)-catenin, or lack of activation of mitogen activated protein kinase (MAPK). Treatment of HT29 cells with phorbol 12-myristate, 13-acetate (PMA), but not arachidonic acid, restored the ability of the cells to spread on laminin in an integrin-dependent manner. Moreover, the addition of both PMA and HGF restored the ability of these cells to scatter on laminin in a synergistic manner. This event correlated with increased tyrosine phosphorylation of paxillin and activation of MAPK. Moreover, when the MEK (MAPK kinase)/MAPK pathway was blocked by the MEK inhibitor PD098059, HGF-induced scattering of HT29 cells was blocked. Thus, HGF modulation of HT29 cell motility is regulated by both integrin and growth factor-dependent signaling and implicates MAPK in the modulation of intercellular adhesion and epithelial cell motility.

Actins↗
PubMed1986

n-Butyrate alters chromatin accessibility to DNA repair enzymes.

Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

Butyrates↗
PubMed2006

Specific detection of cytokeratin 20-positive cells in blood of colorectal and breast cancer patients by a high sensitivity real-time reverse transcriptase-polymerase chain reaction method.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

Biomarkers, Tumor↗
PubMed1994

Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells.

The polar-planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage-independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation. HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short- and long-term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down-regulation of c-myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane-associated alkaline phosphatase activity, cytosolic mucin content, PAS+/diastase-resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype.

Acetamides↗