Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

Atypical microtubule organization in undifferentiated human colon cancer cells

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.     

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub-domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non-homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.     

Antiproliferative effect of DNA polymerase alpha antisense oligodeoxynucleotides on breast cancer cells

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase alpha (pol alpha) plays a relevant role in DNA synthesis and cell proliferation. Pol alpha gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer pol alpha antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 microM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and cell count. [3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of pol alpha mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of pol alpha in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.     

Effect on colon cancer cells of human interferon-beta gene entrapped in cationic multilamellar liposomes

When cultured cells of human colon cancer cell line SW480 were transfected with human interferon-beta (hIFN-beta) gene by means of cationic multilamellar liposomes, the endogenously produced hIFN-beta exhibited a remarkable anti-proliferative effect on the cells, which was more effective than that of exogenously added hIFN-beta. This effect lasted for several days, and was blocked completely by the addition of sufficient amounts of anti-hIFN-beta antibody. From experiments using a transwell plate and an infusion pump, we found that endogenously produced hIFN-beta acted effectively on the cells around the transfectants and that the growth-inhibitory effect was totally retained upon continuous dilution of the medium. These data indicate that hIFN-beta expressed endogenously by transfer of its gene acted on these cancer cells mainly in a paracrine manner. Although the transfection with hIFN-gamma gene also revealed a definite growth-inhibitory effect on the same tumor cells, the extent was less than that of hIFN-beta gene.     

Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells

Mucinous colorectal cancers have a poorer prognosis than colorectal cancers which produce a low amount of mucin, but the exact mechanism is not well understood. The present study was undertaken to elucidate the possible mechanisms of invasion and metastasis of colon cancer cells producing high levels of mucin using mucin glycosylation inhibitor, benzyl-alpha-N-acetylgalactosamine. The binding activity of treated HM7 cells to endothelial leukocyte adhesion molecule (ELAM-1) was significantly decreased and fixed cell binding of MoAb SNH-3 and 19-9 (specific for sialyl Le(x) and sialyl Le(a), respectively) was also significantly decreased. Metalloproteinase activity in conditioned medium and invasion of matrigel-coated porous filters by treated HM7 cells were decreased. However, there was no difference between control and treated HM7 cells in terms of matrix protein binding. These results suggest that O-glycosylated mucin is important in the invasive and metastatic properties of HM7 human colon cancer cells.     

Effects of 5'-aminothymidine and leucovorin on radiosensitization by iododeoxyuridine in human colon cancer cells

We examined the effects of two biochemical modulators, 5'-aminothymidine (5'-AdThd) and leucovorin (LV), on the in vitro incorporation of iododeoxyuridine (IdUrd) into DNA and its subsequent radiosensitization in two human colon cancer cell lines, HT 29 and HCT 116. 5'-AdThd is a modulator of thymidine kinase activity while LV is an essential cofactor for thymidylate synthase activity. In HT 29 cells, the combination of 5'-AdThd (10 &mgr;m) and IdUrd (1-10 &mgr;m) resulted in a significant increase in IdUrd triphosphate pools and in IdUrd-DNA incorporation. Coadministration of LV (10 &mgr;m) with IdUrd (1-10 &mgr;m) resulted in a significant decrease in thymidine triphosphate pools and a comparable increase in IdUrd-DNA incorporation as the combination of 5'-AdThd + IdUrd. The increase in radiosensitization by clonogenic survival with either combination was a direct linear function with the percentage of IdUrd-DNA incorporation. For HCT 116 cells, however, the results were different. While 5'-AdThd + IdUrd resulted in an increase in IdUrd triphosphate and percentage of IdUrd-DNA incorporation, significant cytotoxicity was noted. The radiosensitivity of HCT 116 cells treated with 5'-AdThd + IdUrd was not a linear function above 25% IdUrd-DNA incorporation. Also, no increase in IdUrd-DNA incorporation or radiosensitization was observed with LV + IdUrd although LV enhanced the decrease in thymidine triphosphate pools by IdUrd treatment. These results indicate heterogeneity in the response of different colon cancer cells to these modulators which may be related to the regulation of deoxynucleotide metabolic enzymes.     

Wild-type p53 protein potentiates cytotoxicity of therapeutic agents in human colon cancer cells

Wild-type p53 is induced by DNA damage. In different cell types, this induction is suggested either to facilitate DNA repair by inducing a cell cycle pause or to potentiate cell death via apoptosis. Wild-type p53 in different cell types has similarly been associated with either enhancement of or increased resistance to the cytotoxicity of many cancer therapeutic agents. We have constructed a colorectal cancer cell line bearing, in addition to endogenous mutant p53 alleles, an exogenous wild-type p53 allele that is under the regulatable control of the lac repressor. Induction of wild-type p53 by isopropyl-beta-thiogalactopyranoside in these cells induces a reversible growth arrest but does not induce cell death. However, we find that the induction of wild-type p53 powerfully potentiates the cytotoxicity of both irradiation and 5-fluorouracil, two agents that are used clinically in the treatment of colorectal cancer. We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer. These findings suggest that the common loss of wild-type p53 in many colorectal cancers may play a role in the clinical resistance of these tumors to anticancer agents. Although some cancer cells may not be directly killed by p53 gene therapy, our findings suggest that genetic alteration of some cancers to induce wild-type p53 may increase their sensitivity to cytotoxic gene therapy.     

Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells

BACKGROUND & AIMS: The epidermal growth factor receptor (EGFR) is under investigation as a therapeutic target for cancers. Colon cancer cell lines are variably dependent on autocrine stimulation of EGFR. We therefore examined the effects of a selective EGFR tyrosine kinase inhibitor, PD 153035, on proliferation and survival of five colon cancer cell lines whose autonomous proliferation is either EGFR ligand dependent or EGFR ligand independent. METHODS: Effects of inhibitors were screened by MTS growth assays, [3H]thymidine incorporation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence microscopy, immunoblotting, and in vitro protease assays. RESULTS: PD 153035 caused dose-dependent cytostasis (200 nmol/L to 1 micromol/L) and apoptosis (>10 micromol/L) in ligand-dependent cell lines and caused variable apoptosis (>10 micromol/L) but no cytostasis in ligand-independent cell lines. Apoptosis induced by 10 micromol/L PD 153035 was not associated with induction of p53 protein expression but was accompanied by activation of caspases that cleave poly(ADP-ribose) polymerase, lamin B1, and Bcl-2. Inhibition of caspase 3-like protease activity by DEVD-fluoromethylketone significantly delayed the onset of PD 153035-induced apoptosis. CONCLUSIONS: The EGFR tyrosine kinase inhibitor PD 153035 induces cytostasis and caspase-dependent apoptosis in EGFR ligand-dependent colon cancer cell lines. These observations encourage further investigation of EGFR tyrosine kinase inhibitors for treatment of colorectal neoplasms.     

Are dietary factors involved in DNA methylation associated with colon cancer?

Albuterol is a quickly acting beta-2 adrenergic agonist bronchodilator widely used by asthmatics. Because recent case-control studies have suggested a relationship between the increase in mortality of asthmatics over the past decade and the use of beta 2-adrenergic agonists in the control of asthma, concern has developed regarding the potential cardiotoxicity of beta 2-specific adrenergic agonists, including albuterol. The aim of this investigation was to assess the potential for cardiotoxicity of inhaled albuterol dry powder in rats, monkeys, and dogs. All species were exposed to an aerosol of albuterol 1 h per day, 7 days per week, for at least 2 weeks. Control groups were exposed to filtered conditioned air and handled in the same manner as the albuterol-exposed animals. Plasma concentrations of albuterol confirmed systemic exposure. The daily inhaled dose received by the animals was calculated based on measured respiratory minute volumes, published respiratory tract deposition data, as well as HPLC-determined particle size distribution data and aerosolized albuterol concentrations. Multiples of the maximum daily clinical dose (presentation of 15 micrograms/kg in a 70-kg human) were approximately 0.25- to 2500-fold in the rat, 9- to 100-fold in the monkey, and 0.5- to 90-fold in the dog. No findings attributed to albuterol were observed in the monkey. Tachycardia and transient hypokalemia occurred in rats at multiples of 1.5 times or greater of the maximum clinical dose. Absolute and relative heart weights increased in rats receiving multiples of 47 times or greater of the maximum human dose. In the absence of histopathologic findings, the increases in rat heart weights were considered a physiologic hypertrophic response to tachycardia. In dogs tachycardia and transient hypokalemia occurred at all doses tested. Slight to mild fibrosis in the papillary muscles of the left ventricle of the heart occurred in dogs at multiples > or = 19 times the clinical dose. The cardiovascular effects observed were consistent with the known pharmacologic action of beta 2-adrenergic agonists. Due to the lack of toxicologically relevant findings in rats and monkeys and the wide safety margin in dogs, the findings in this study do not suggest a cardiotoxicity risk in the human population after repeated exposures to clinical doses of albuterol currently used in the treatment of asthma.     

The nonfermentable dietary fiber lignin alters putative colon cancer risk factors but does not protect against DMH-induced colon cancer in rats

The effect of supplementation of the diet with autohydrolyzed lignin on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 112 male Sprague-Dawley rats. Rats received eight weekly injections of DMH (9.5 mg/kg s.c.) or the saline vehicle solution and then were maintained on a basal AIN-76 fiber-free diet or the basal fiber-free diet plus 5% or 10% (wt/wt) lignin for 24 weeks. Rats were killed 32 weeks after the start of the experiment. Colon tumor incidence, location, and multiplicity were determined. Body weight, caloric intake, fecal dry weight, gut transit time, pH of cecal contents, and total fecal bile acid excretion were measured. Supplementation of the diet with 5% or 10% lignin resulted in increased fecal dry weight and total fecal bile acid excretion and in decreased gut transit time, colon pH, and fecal bile acid concentration. Dietary lignin did not significantly affect colon tumor incidence or multiplicity compared with the fiber-free diet. Thus dietary supplementation with autohydrolyzed lignin, a food fiber with good bulking characteristics, had a significant effect on several factors that have previously been linked to reduction of colon cancer risk, but the consumption of high levels of lignin did not decrease the risk for colon cancer.     

Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.     

Possible effect of pneumoperitoneum on the spreading of colon cancer tumor cells

Development of the central somatosensory system is profoundly modulated by the sensory periphery. Cauterization of facial whiskers alters the segregation pattern of barrels in rodents only during a few days just after birth (critical period). Although a molecular basis of the segregation of barrel neurons and the critical period for the anatomical plasticity observed in layer IV barrel neuron is not clear yet, the accumulating evidence suggests that neurotrophins modulate synaptic connections including central nervous system. In this study, we showed by in situ hybridization that mouse barrel side neurons express brain-derived neurotrophic factor (BDNF) mRNA and both catalytic and non-catalytic forms of trkB mRNA. Cautery of row C vibrissae on the right side of the face within 24 h after birth (post natal day 0, PND0) reduced the expression of BDNF and trkB mRNA from the division region between the contralateral row C barrels at PND7. The vibrissae in row A, C, and E were cauterized at PND0 followed by quantitative RT-PCR for BDNF and trkB mRNA with total RNA isolated from the barrel region at PND7. The result showed that BDNF, but not trkB, mRNA was increased several-fold in the contralateral barrel region. These data suggest that the expression of BDNF mRNA is differentially regulated between injured barrels and actively innervated barrels. The differential expression of the mRNA encoding neurotrophins and their receptors may be important in regulating the injury-dependent re-segregation of barrels.     

Preferential antibody recognition of structurally distinct HIV-1 gp120 molecules

We have developed an assay, using a biosensor matrix and surface plasmon resonance, that rapidly and reproducibly measures antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp120 in various structural conformations. In particular, antibodies displaying preferential reactivity to a CD4-binding competent ("native," rgp120) or CD4-binding incompetent ("reduced," rcmgp120) monomeric gp120 molecule were distinguished. This technique has advantages over conventional enzyme-linked immunosorbent assay (ELISA) methodology in which it is difficult to control the concentration of protein adsorbed to the ELISA wells and a significant disruption of protein structure occurs on adsorption. A population of gp120 molecules that lacked CD4 receptor binding capacity and bound antibodies specific for reduced gp120 was found in several native gp120 preparations. The relative amount of this CD4-binding incompetent population varied among the various preparations studied. This presence of CD4-binding incompetent molecules within various native recombinant gp120 preparations may have implications for HIV-1 envelope vaccine development. By measuring antibody-binding ratios, several monoclonal antibodies were identified, which, although elicited by immunization with various native gp120 preparations, bound specifically to reduced gp120. The ability to screen antibody specificity against HIV-1 envelope proteins with different conformations will assist in determining the quality of antibodies induced by various HIV-1 envelope vaccine candidates.     

Hepatocyte extracellular matrix modulates expression of growth factors and growth factor receptors in human colon cancer cells

A new algorithm for analysis of the homology and genetic semihomology in protein sequence is described. It assumes the close relation between the compared amino acids and their codons in related proteins. The algorithm is based on the network of the genetic relationship between amino acids and, thus differs from the commonly used statistical matrices. The results obtained by using this method are more comprehensive than used at present, and reflect the actual mechanism of protein differentiation and evolution. They concern: (1) location of homologous and semihomologous sites in compared proteins; (2) precise estimation of insertion/deletion gaps in non-homologous fragments; (3) analysis of internal homology and semihomology; (4) precise location of domains in multidomain proteins; (5) estimation of genetic code of non-homologous fragments; (6) construction of genetic probes; (7) studies on differentiation processes among related proteins; (8) estimation of the degree of relationship among related proteins; (9) studies on the evolution mechanism within homologous protein families and (10) confirmation of actual relationship of sequences showing low degree of homology.     

Integrin-mediated signalling of gelatinase B secretion in colon cancer cells

The progression of colon cancer has been linked to both cell adhesion molecules called integrins and matrix-degrading enzymes called metalloproteinases. Herein we report that the alpha v beta 6 integrin expressed in colon cancer cells induces gelatinase B secretion through the C-terminal cytoplasmic extension unique to the beta 6 integrin subunit, and that this ligand-independent event involves activation of the protein-kinase-C pathway.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cells growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were a completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.