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PubMed1991

Different effects of sequential combinations of N-methylformamide with 5-fluorouracil on human colon carcinoma cells growing in nude mice.

The effects of the combination of N-methylformamide (NMF) with 5-fluorouracil (5-FU) on tumor growth and morphological features of human colon carcinoma cells (HT29) implanted in nude mice were assessed. Both agents were administered i.p. at tolerable doses: 5-FU at 19 mg/kg for 5 days and NMF at 200 mg/kg for 12 days. Four main schedules were tested: 5-FU alone, NMF alone, NMF followed by 5-FU and 5-FU followed by NMF. The last sequence was the most effective, as compared with the other treatment regimens. In particular, the 5-FU----NMF combination induced a tumor inhibition of about 75% at the end of the treatments (17th day) versus an inhibition of 23%-43% in the other schedules. Morphological observations, carried out by light and electron microscopy, indicated a possible relationship between the presence of structural changes and tumor growth inhibition. The results of this study renew interest in the use of NMF in sequential combination confirming sequence as a critical factor for the optimal combination of NMF and 5-FU.

Adenocarcinoma↗
PubMed1988

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells.

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.

Adenocarcinoma↗
PubMed2005

Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutathione and the modulation of glutathione S-transferase A4 expression by butyrate.

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.

Aldehydes↗
PubMed1997

Combination therapy with 5-fluorouracil and IFN-alpha2a induces a nonrandom increase in DNA fragments of less than 3 megabases in HT29 colon carcinoma cells.

We have used pulsed-field gel electrophoresis to examine 5-fluorouracil (5FU)-induced DNA double-strand breaks (DSBs), both with and without modulation by IFN-alpha2a (IFNalpha), in HT29 human colon adenocarcinoma cells. Although 24-h treatment with either 10 microM 5FU or 500 units/ml IFNalpha did not result in significant DNA fragmentation, the combination of 5FU + IFNalpha resulted in a significant increase in DNA DSBs versus either drug alone (P < 0.05). The pattern of fragmentation induced by treatment with 5FU + IFNalpha was compared to that induced by gamma-radiation, which generates lesions at random sites, digestion with NotI restriction endonuclease, which cleaves at the specific sequence 5' ellipsis GCGGCCGCellipsis 3', and HhaI restriction endonuclease, which cleaves at the specific sequence 5'ellipsis GCGCellipsis 3'. 5FU + IFNalpha resulted in a specific pattern characterized by the accumulation of fragments of <3 Mb in the absence of fragments of >3 Mb, which differed from that of gamma-radiation and restriction endonuclease digestion. Because neither morphological nor DNA fragmentation characteristic of apoptosis was observed after 5FU + IFNalpha treatment, the nonrandom pattern of DSBs that was observed did not appear to be the result of the initiation of programmed cell death within these cells.

Apoptosis↗
PubMed2000

IkappaBbeta-related proteins in normal and transformed colonic epithelial cells.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that can influence cell proliferation, apoptosis, and inflammatory responses. Since these events can contribute to carcinogenesis, we examined the expression of NF-kappaB inhibitory proteins (IkappaBs) in normal and transformed colonic epithelial cells. Immunohistochemical analysis of the mouse colon revealed a high level of IkappaBbeta expression in epithelial cells relative to the rest of the tissue, whereas IkappaBalpha was found primarily in cells of the lamina propria. Mouse colon tumors showed a similar cell-specific staining pattern. Immunoblot analysis of IkappaBbeta from mouse colonocytes and the human HT-29 colon cancer cell line indicated that most of the IkappaBbeta in these cells was similar to the C-terminal-truncated IkappaBbeta2 isoform. Cell fractionation studies were consistent with IkappaBbeta being a major regulator of p65-p50 NF-kappaB complexes in HT-29 cells. Interestingly, two larger proteins specifically recognized by IkappaBbeta antibodies (p106 and p112) were found in HT-29 cells and in colon tissue of carcinogen-exposed mice. The p106 and p112 proteins bound to NF-kappaB, and their levels changed during the transient interleukin-1beta activation of NF-kappaB in HT-29 cells. Evidence was obtained indicating that p106 and p112 are stably ubiquitinated forms of IkappaBbeta. We propose that deficiencies in the proteasomal degradation of IkappaBbeta lead to p106 and p112 accumulation, which in turn alter NF-kappaB regulation in colon cancer cells.

Animals↗
PubMed2003

Analysis of genetic stability at the EP300 and CREBBP loci in a panel of cancer cell lines.

EP300 (p300) and CREBBP (CBP) are highly related transcriptional co-activators possessing histone acetyltransferase activity. These proteins have been implicated in coordinating numerous transcriptional responses that are important in the processes of proliferation and differentiation. A role for EP300 and CREBBP as tumor suppressors in cancer has been suggested by the fact that they are targeted by viral oncogenes; there is an increased incidence of hematologic malignancies in mice monoallelic for CREBBP; and loss, albeit at a low frequency, of both EP300 alleles in epithelial cancers has been observed. Because the level of EP300/CREBBP appears to have a critical effect on integrating certain transcriptional processes, we sought to determine whether a loss in the combined gene dosage of EP300 and CREBBP might play a role in cancer. Accordingly, we screened a panel of 103 cell lines for loss of heterozygosity and found 35 and 51% LOH for the CREBBP and EP300 loci, respectively. Concordant loss of CREBBP and EP300 was not associated with mutations in important regions of the remaining EP300 or CREBBP genes. In addition, there did not appear to be a statistically significant selection in cancer cells, stratified by various criteria, for the concordant loss of EP300 and CREBBP. We conclude that EP300 and CREBBP rarely act as classical tumor suppressors in human cancer.

CREB-Binding Protein↗
PubMed2004

Mapping of a candidate colorectal cancer tumor-suppressor gene to a 900-kilobase region on the short arm of chromosome 8.

Loss of heterozygosity (LOH) on 8p occurs at high frequencies in many tumor types, including colorectal carcinoma (CRC). We previously used microcell-mediated chromosome transfer (MMCT) into the CRC cell line SW620 to map a approximately 7.7-Mb colorectal cancer-suppressor region (CRCSR) at 8p22-23.1. In the current study, we transferred small fragments of this CRCSR into SW620 to refine the region further. Two microcell hybrids containing a 321- to 898-kb region around the D8S552 marker at 8p23.1 showed suppression of soft agar clonicity and tumorigenicity in athymic mice when compared to control cell lines. These data suggest that the putative colorectal tumor-suppressor gene is within this small region. We analyzed two candidate genes within this region: FLJ23749 and KIAA1456. Expression of both genes was detected in normal colonic crypt cells and in mucosa. Quantitative reverse transcriptase polymerase chain reaction showed downregulation of KIAA1456 in 9 of 12 primary colorectal tumors compared to matching normal mucosa, but normal or increased expression of FLJ23749. FLJ23749 was expressed in all CRC cell lines tested; however, KIAA1456 was downregulated in three cell lines, including SW620, and was restored in the suppressed microcell hybrids. 5'aza-2'Deoxycytidine treatment of the downregulated cell lines restored expression of KIAA1456, but bisulfite genomic sequencing did not show a correlation between promoter methylation and expression. Forty percent of the primary tumors showed LOH at this CRCSR locus, and mutation analysis revealed somatic mutations in 1 of 88 primary colorectal tumors for both KIAA1456 and FLJ23749. Despite the rarity of somatic mutations, the expression data suggest that KIAA1456 is still a candidate for the putative 8p colorectal cancer tumor-suppressor gene.

Animals↗
PubMed2006

Ursodeoxycholic acid modulates histone acetylation and induces differentiation and senescence.

Agents that can modulate colonic environment and control dysregulated signaling are being evaluated for their chemopreventive potential in colon cancer. Ursodeoxycholate (UDCA) has shown chemopreventive potential in preclinical and animal models of colon cancer, but the mechanism behind it remains unknown. Here biological effects of UDCA were examined to understand mechanism behind its chemoprevention in colon cancer. Our data suggests that UDCA can suppress growth in a wide variety of cancer cell lines and can induce low level of apoptosis in colon cancer cells. We also found that UDCA treatment induces alteration in morphology, increased cell size, upregulation of cytokeratin 8, 18 and 19 and E-cadherin, cytokeratin remodeling and accumulation of lipid droplets, suggesting that UDCA induces differentiation in colon carcinoma cells. Our results also suggest significant differences in UDCA and sodium butyrate induced functional differentiation. We also report for the first time that UDCA can induce senescence in colon cancer cells as assessed by flattened, spread out and vacuolated morphology as well as by senescence marker beta-galactosidase staining. We also found that UDCA inhibits the telomerase activity. Surprisingly, we found that UDCA is not a histone deacytylase inhibitor but instead induces hypoacetylation of histones unlike hyperacetylation induced by sodium butyrate. Our results also suggest that, although UDCA induced senescence is p53, p21 and Rb independent, HDAC6 appears to be important in UDCA induced senescence. In summary, our data shows that UDCA modulates chromatin by inducing histone hypoacetylation and induces differentiation and senescence in colon cancer cells.

Acetylation↗
PubMed2006

Cell differentiation dependent expressed CCR6 mediates ERK-1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells.

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

Caco-2 Cells↗
PubMed2007

NF-kappaB pathway is involved in griseofulvin-induced G2/M arrest and apoptosis in HL-60 cells.

Griseofulvin (GF), an oral antifungal agent, has been shown to exert antitumorigenesis effect through G2/M cell cycle arrest in colon cancer cells. But the underlying mechanisms remained obscure. The purpose of this study is to test the cytotoxic effect of GF on HL-60 and HT-29 cells and elucidate its underlying molecular pathways. Dose-dependent and time-course studies by flow cytometry demonstrated that 30 to 60 microM GF significantly induced G2/M arrest and to a less extend, apoptosis, in HL-60 cells. In contrast, only G2/M arrest was observed in HT-29 cells under similar condition. Pretreatment of 30 microM TPCK, a serine protease inhibitor, completely reversed GF-induced G2/M cell cycle arrest and apoptosis in HL-60 cells but not in HT-29 cells. The GF-induced G2/M arrest in HL-60 cells is reversible. Using EMSA and super-shift analysis, we demonstrated that GF stimulated NF-kappaB binding activity in HL-60 cells, which was completely inhibited by pretreatment of TPCK. Treatment of HL-60 with 30 microM GF activated JNK but not ERK or p38 MAPK and subsequently resulted in phosphorylation of Bcl-2. Pretreatment of TPCK to HL-60 cells blocked the GF-induced Bcl-2 phosphorylation but not JNK activation. Time course study demonstrated that activation of cdc-2 kinase activity by GF correlated with Bcl-2 phosphorylation. Taken together, our results suggest that activation of NF-kappaB pathway with cdc-2 activation and phosphorylation of Bcl-2 might be involved in G2/M cell cycle arrest in HL-60 cells.

Antifungal Agents↗
PubMed1992

Modulation of gene expression as a biomarker in colon.

Computer-driven scanning and image processing methodology has demonstrated that genetic inheritance of risk for colorectal cancer in familial polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) families is associated with highly pleiotropic effects on patterns of gene expression in the flat colonic mucosa. The mitochondrial (mt) gene encoding subunit 3 of cytochrome oxidase (COXIII) is one of a panel of cloned sequences which characterize genetic risk. Expression of COXIII decreased in progression of, and risk for, colonic tumors in vivo. Further, metabolizable, unbranched, short-chain fatty acids (SCFAs) elevated expression of mtCOXIII, as well as mtCOXI, in HT29 cells and also elevated mtCOX enzymatic activity. However, expression of nuclear encoded COX subunits were unaffected. These changes may be related to documented alterations in mitochondria structure and function in transformed colonic epithelial cells. SCFAs produced when colonic microflora causes fermentation of fiber are the principle energy source for normal colonic epithelial cells; SCFAs also induce a more differentiated phenotype both in vitro and in vivo. Therefore, a mechanistic link may exist between molecular events in inherited risk and a dietary factor (fiber) which may modulate such risk. In a preliminary intervention trial in collaboration with M. Lipkin, high risk HNPCC patients received daily supplements of 1500 mg CaCO3 per day, which may be protective for development of colorectal tumors. Elevations in COXIII expression were seen in 7 of 12 patients within the first 7 months, followed by complex changes in expression of this sequence.

Adenomatous Polyposis Coli↗
PubMed2006

Repression of HIP/RPL29 expression induces differentiation in colon cancer cells.

We had previously shown that the expression of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) was upregulated in colon cancer tissues. The present study investigated the role of HIP/RPL29 in differentiation in colon cancer cells. Inducing cellular differentiation in HT-29 cells by both sodium butyrate and glucose deprivation resulted in a significant downregulation of HIP/RPL29 expression. The beta-catenin/Tcf-4 pathway is the most important pathway controlling the switch between cellular differentiation and proliferation in intestinal epithelial cells. Inducing differentiation by dominant-negative inhibition of the beta-catenin/Tcf-4 complexes in LS174T cells also resulted in downregulation of HIP/RPL29. To determine whether a lower expression of HIP/RPL29 could induce differentiation in cancer cells, small interfering RNA (siRNA) targeting HIP/RPL29 was transfected into LS174T cells. The resultant knockdown of HIP/RPL29 expression induced cellular differentiation, as shown by the increased expression of two known markers of differentiation in LS174T cells, galectin-4 and mucin-2. In addition, the differentiation process induced by repression of HIP/RPL29 expression was accompanied by the upregulation of p21 and p53. In conclusion, HIP/RPL29 plays a role in the cellular differentiation process in colon cancer cells. The differentiation process is at least partially mediated by the upregulation of p21 and p53 pathways.

Alkaline Phosphatase↗
PubMed1998

Isoelectrofocusing phenotype and relative concentration of transcobalamin II isoproteins related to the codon 259 Arg/Pro polymorphism.

We investigated transcobalamin II (TC) isoelectrofocusing (IEF) phenotype and codon 259 polymorphism, in Caco-2 and HT-29 cells and in blood drawn from 39 healthy Caucasians. Caco-2 cells expressed a single TC variant (259-Arg), while HT-29 cells expressed TC with either Arg or Pro at codon 259 and exhibited two isoproteins in IEF with urea, but only one in IEF without urea. Among the Caucasians, 7 subjects expressed the TC 259-Arg variant, 10 the 259-Pro variant, and 22 were heterozygous. The TC 259-Pro isoprotein issued from HT-29 cells and heterozygous caucasian sera, was, respectively, 2. 4-fold and 1.6-fold higher than the TC 259-Arg isoprotein. Apo-TC and vitamin B12 serum concentrations in 259-Pro homozygotes were, respectively, 1.7 and 1.4-fold higher than those in 259-Arg homozygotes (p<0.005 and p=0.05). In conclusion, the 259-Arg/Pro polymorphism yields two TC variants only titratable in denaturing conditions and affects the blood level of both Apo-TC and vitamin B12.

Adult↗
PubMed2001

Syndecan-2 expression in colorectal cancer-derived HT-29 M6 epithelial cells induces a migratory phenotype.

Members of the heparan sulfate proteoglycan family, the syndecans have emerged as integrators of extracellular signals, such as ECM components or growth factors, that activate cytoplasmic signaling cascades and regulate cytoskeletal functions. Specifically, syndecan-2 has been implicated in various cellular processes, from differentiation to migration, including its participation in cell-cell and cell-matrix adhesion. Here, we focused on the involvement of syndecan-2 in epithelial versus mesenchymal differentiation. Colorectal cancer-derived HT-29 M6 epithelial cells were stably transfected with full-length syndecan-2 cDNA, and the effect on cell morphology, adhesion, and mobility was evaluated. Characteristic features of migratory cells such as loss of intercellular contacts, flatter shape and multiple membrane projections were observed in syndecan-2 transfectants. Western blot analysis of the major component of epithelial adherens junctions, E-cadherin, revealed decreased expression levels. Furthermore, syndecan-2 induced stronger adhesion to collagen type I, specifically inhibited by heparin. This was correlated with an increased ability for migration, as demonstrated by wound healing experiments and transwell assays, without affecting their growth rate. These results indicate that syndecan-2 expression in mucus-secreting HT-29 M6 cells induces differentiation toward a migratory mesenchymal-like phenotype.

Adenocarcinoma↗
PubMed1998

Regulation of human lymphocyte IL-4 secretion by intestinal epithelial cell-derived interleukin-7 and transforming growth factor-beta.

A mucosal immune response to food antigens could result in detrimental hypersensitivity responses. Therefore, the response to many orally administered antigens is downregulated by mechanisms which are not completely understood. Intestinal epithelial cells (IEC) in these tissues may play a role in these regulatory mechanisms via their secreted cytokines. Experiments with human lymphocytes or isolated CD4(+) T cells cultured with 4-day culture supernatants from human colonic carcinoma cell lines revealed that the IEC cell lines normally secreted levels of IL-7 which could enhance IL-4, but not IL-2 or IFN-gamma, secretion by stimulated mixtures of lymphocyes, but not purified CD4(+) T cells. However, acid treatment of the IEC culture supernatants to activate latent TGF-beta resulted in a suppression IL-4, but not IL-2 or IFN-gamma, secretion. These results indicate that under normal conditions, IEC secrete latent TGF-beta and IL-7, the latter of which may enhance local IL-4 secretion. However, activation of the IEC-derived TGF-beta may suppress local IL-4 secretion to suppress the induction of local Th2-type responses to intestinal lumenal antigens.

CD4-Positive T-Lymphocytes↗
PubMed1996

Brefeldin A is a potent inducer of apoptosis in human cancer cells independently of p53.

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 microM. The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.

Alkaloids↗
PubMed1999

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.

Animals↗
PubMed2002

In vitro studies of gadolinium-DTPA conjugated with monoclonal antibodies as cancer-specific magnetic resonance imaging contrast agents.

The monoclonal antibodies, 9.2.27 against human melanoma cell lines and WM53 against leukemia cell lines, were conjugated with cyclic anhydride gadolinium-diethylenetriaminepenta-acetic acid (Gd-cDTPAa) and used as tumor-specific contrast agents in magnetic resonance imaging (MRI). The data indicate that Gd-DTPA-9.2.27 in solution decreased the T1 relaxation of water protons at 7.0 Tesla (300 MHz) in direct proportion to the gadolinium concentration, and this effect was greater than in Gd-DTPA solutions. These conjugates show high specificity for melanoma and leukemia cell lines. T1 relaxation time at 7.0 Tesla, measured for the melanoma cell line (MM-138) and leukemia cell line (HL-60) after incubation at 37 degrees C for 4 hr, were significantly decreased (approximately 25%) relative to controls. The gadolinium concentration in cells and washing solutions was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). A linear relationship was observed between T1 relaxation rates and gadolinium concentrations obtained by ICP-AES. The ICP-AES results showed no gadolinium uptake in the non-targeted HT-29 colorectal cancer cells.

Animals↗