agar and Colonic-Neoplasms

Topics: Agar; Benzhydryl Compounds; beta Catenin; Colonic Neoplasms; Fluorouracil; HCT116 Cells; HSP27 Heat-Shock Proteins; Humans; Mucous Membrane; Phenols; Ribosomal Protein S6 Kinases, 70-kDa; Tumor Suppressor Protein p53

Numerous health benefits of diets containing red seaweeds or agar-derived sugar mixtures produced by enzymatic or acid hydrolysis of agar have been reported. However, among various agar-derived sugars, the key components that confer health-beneficial effects, such as prebiotic and anti-colon cancer activities, remain unclear. Here, we prepared various agar-derived sugars by multiple enzymatic reactions using an endo-type and an exo-type of β-agarase and a neoagarobiose hydrolase and tested their in vitro prebiotic and anti-colon cancer activities. Among various agar-derived sugars, agarotriose exhibited prebiotic activity that was verified based on the fermentability of agarotriose by probiotic bifidobacteria. Furthermore, we demonstrated the anti-colon cancer activity of 3,6-anhydro-l-galactose, which significantly inhibited the proliferation of human colon cancer cells and induced their apoptosis. Our results provide crucial information regarding the key compounds derived from red seaweeds that confer beneficial health effects, including prebiotic and anti-colon cancer activities, to the host.

Topics: Agar; Antineoplastic Agents; Apoptosis; Bifidobacterium; Cell Proliferation; Colonic Neoplasms; Fermentation; Galactose; HCT116 Cells; Humans; Hydrolysis; Prebiotics; Rhodophyta; Seaweed

Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel. These protocols can also be used to conduct functional cDNA or CRISPR screens for modulators of anchorage-independent growth.

Topics: Agar; Cell Adhesion; Colonic Neoplasms; Gene Silencing; High-Throughput Screening Assays; Humans; Neoplasm Proteins; RNA, Small Interfering; Tumor Cells, Cultured

More than 80 years ago Otto Warburg suggested that cancer might be caused by a decrease in mitochondrial energy metabolism paralleled by an increase in glycolytic flux. In later years, it was shown that cancer cells exhibit multiple alterations in mitochondrial content, structure, function, and activity. We have stably overexpressed the Friedreich ataxia-associated protein frataxin in several colon cancer cell lines. These cells have increased oxidative metabolism, as shown by concurrent increases in aconitase activity, mitochondrial membrane potential, cellular respiration, and ATP content. Consistent with Warburg's hypothesis, we found that frataxin-overexpressing cells also have decreased growth rates and increased population doubling times, show inhibited colony formation capacity in soft agar assays, and exhibit a reduced capacity for tumor formation when injected into nude mice. Furthermore, overexpression of frataxin leads to an increased phosphorylation of the tumor suppressor p38 mitogen-activated protein kinase, as well as decreased phosphorylation of extracellular signal-regulated kinase. Taken together, these results support the view that an increase in oxidative metabolism induced by mitochondrial frataxin may inhibit cancer growth in mammals.

Topics: Aconitate Hydratase; Adenosine Triphosphate; Agar; Animals; Cell Line, Tumor; Cell Proliferation; Cell Respiration; Colonic Neoplasms; Energy Metabolism; Frataxin; Gene Expression Regulation, Neoplastic; Humans; Intracellular Membranes; Iron-Binding Proteins; Mitochondria; Neoplasm Transplantation; Neoplasms; Oxygen; Oxygen Consumption; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Time Factors; Transfection

HER-2 is constitutively activated and overexpressed in many cancers, and its inhibition in colon cancer cells diminishes tumorigenicity and induces apoptosis. Little is known about the regulation of HER-2 signaling in colon cancer cells. Integrin alpha5/beta1 expression is frequently lost in colorectal cancer cells compared with normal intestinal epithelium, and colon cancer cells lacking integrin alpha5/beta1 expression utilize HER-2 signaling for proliferation and tumorigenicity. Re-expression of integrin alpha5/beta1 in colon cancer cells abrogated their tumorigenicity, but how this occurs is not well known. Stable expression of integrin alpha5/beta1 in colon cancer cells with little or no detectable integrin alpha5/beta1 protein expression resulted in the post-transcriptional down-regulation of HER-2 protein. Integrin alpha5/beta1 was found to interact with HER-2, and the cytoplasmic domain of integrin alpha5/beta1 was sufficient to mediate HER-2 down-regulation. Integrin alpha5/beta1-mediated down-regulation of HER-2 was the result of increased lysosomal targeting. The inhibition of HER-2 signaling represents a potential mechanism by which integrin alpha5/beta1 exerts its tumor suppressor-like activity in colon cancer cells. These results also suggest that a novel function for integrin alpha5/beta1 is the control of HER-2 expression.

Topics: Agar; Biotinylation; Blotting, Northern; Caco-2 Cells; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Colonic Neoplasms; Cytoplasm; Down-Regulation; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Immunoblotting; Immunoprecipitation; Integrin alpha5beta1; Lysosomes; Mutagenesis, Site-Directed; Phosphorylation; Protein Structure, Tertiary; Receptor, ErbB-2; RNA Processing, Post-Transcriptional; RNA, Messenger; Signal Transduction; Time Factors; Transcription, Genetic; Transfection

The cyclin-dependent kinase inhibitor p21cip1/waf1 negatively regulates the progression of cell cycle and the potential usefulness of p21cip1/waf1 gene is proposed in gene therapy. However, studies have demonstrated a protective role of p21cip1/waf1 against apoptosis and little is known about effects of ectopic expression of p21cip1/waf1 on differentiation of colon cancer cells. In the present study, we found diffuse p21cip1/waf1 expression in only a few clinical samples of colorectal cancer with wild-type p53 gene. To explore the role of p21cip1/waf1 in cell growth, apoptosis and differentiation, we constitutively overexpressed p21cip1/waf1 in HT29 colon carcinoma cells. Ectopic overexpression of p21cip1/waf1 was associated with inhibition of CDK2-associated kinase activity, indicating the functionality of the introduced p21cip1/waf1 gene. Overexpression of p21cip1/waf1 caused an appreciable growth inhibition in monolayer and soft agar cultures and it significantly reduced sodium butyrate- but not 5-fluorouracil-induced apoptosis. p21cip1/waf1 overexpressing cells exhibited marked decrease of intestinal differentiation when assayed with intestinal alkaline phosphatase. Our findings suggest that introduction of p21cip1/waf1 gene into colon cancer cells may be useful for inhibiting cell growth but caution should be taken regarding the increased resistance to certain apoptosis-inducing agents and dysregulation of endogenous p21cip1/waf1-mediated differentiation process.

Topics: Agar; Alkaline Phosphatase; Apoptosis; Blotting, Western; Butyrates; Carcinoma; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Mutational Analysis; Flow Cytometry; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Isobutyrates; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Time Factors; Tumor Suppressor Protein p53

The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase.. The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided.. Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80-90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor alpha of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474.. Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.

Topics: Adenocarcinoma; Agar; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Membrane; Cetuximab; Colonic Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Female; Flow Cytometry; Humans; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Piperidines; Precipitin Tests; Quinazolines; Receptors, Vascular Endothelial Growth Factor; Time Factors

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.

Topics: Agar; Animals; Cell Division; Colonic Neoplasms; Culture Media; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neurotensin; Polymerase Chain Reaction; Pyrazoles; Quinolines; Receptors, Neurotensin; Transplantation, Heterologous; Tumor Cells, Cultured

The three-dimensional growth of cultured tumor cell lines (HT29, a colon adenocarcinoma cell line; M21, a melanoma cell line; KB, a nasopharyngeal carcinoma cell line) has been investigated in an agar culture system with a fibrin matrix in vitro. The tumor cells developed to tumors 3 x 3 mm in diameter after 10 days in culture in vitro. This size was large enough to allow histologic examination. The tumor cells located in the surface area of the three-dimensional tumor seemed to grow well. However, the tumor cells in the center degenerated or did not proliferate, indicating a lack of nutrition and/or anoxia in the center. The histologic comparison between the xenografted tumors on nude mice and the three-dimensional tumors in vitro suggests that the structures of the three-dimensional tumors were comparable, especially in the surface area, to the xenografted tumors. Furthermore, the antitumor effect of mitomycin C on the three-dimensional tumors was found to be dose-dependent.

Topics: Adenocarcinoma; Agar; Animals; Cell Division; Cell Hypoxia; Colonic Neoplasms; Culture Techniques; Extracellular Matrix; Fibrin; Humans; KB Cells; Melanoma; Mice; Mice, Nude; Mitomycin; Transplantation, Heterologous; Tumor Cells, Cultured

Earlier studies showed that the extracellular matrix and the conditioned medium from colon carcinoma support and catalyze the conversion of normal epithelial to colon carcinoma cells (14, 16). Since the cause of this apparent change in malignant potential is completely unknown, the following experiments examined molecular changes accompanying and mediating the transition. Colon epithelial cell cultures were initiated from normal colon biopsies. The cell cultures were carried on in standard medium on fibronectin-coated plates, and in conditioned medium on extracellular matrix from confirmed colon carcinoma. At time intervals, during 12 months period, aliquots were harvested, transferred into roller bottles to obtain enough cells to isolate cellular Poly(A)+-enriched RNA, cell membrane oligosaccharides and to determine cellular growth characteristics. During the 12 months in vitro culture, normal colon epithelial (NCE) cells grown on fibronectin in the standard growth medium maintained their initial characteristics. Whereas, NCE cells grown on the extracellular matrix and in colon carcinoma conditioned medium, progressively acquired the ability to form colonies in soft agar, to form tumors when implanted subcutaneously and to metastasize into the liver when administered intravenously into athymic mice. Poly(A)+-enriched RNA from NCE cells grown on fibronectin and in standard culture medium, did not, whereas the RNA from NCE cells grown on the extracellular matrix and in the colon carcinoma conditioned medium hybridized with 32P-cDNA from colon carcinoma. There were significant changes in the composition and profile of the oligosaccharides from membranes of NCE cells grown on the extracellular matrix. There was significant correlation (P less than 0.001) between the last characteristics.

Topics: Agar; Animals; Cell Membrane; Cells, Cultured; Colon; Colonic Neoplasms; Culture Media; Epithelial Cells; Epithelium; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; Tumor Stem Cell Assay

We studied the factors that control the capacity of tumor cells to grow in soft agar. And, we analyzed the effect of cell-free ascites (CFA) obtained from ovarian cancer patients in combination with various media on the cloning efficiency of H-134 and OVCAR-3nu ovarian carcinoma cell lines and the WiDr colon carcinoma cell line. Seven batches of CFA consistently enhanced the soft agar growth of tumor cells more efficiently than tested sera. The addition of charcoal-treated bovine serum albumin (BSA) lowered the amount of CFA required for optimal tumor cell growth. As little as 1.25 ng of epidermal growth factor (EGF)/ml further improved OVCAR-3nu soft agar growth in combination with all of the amounts of CFA tested. Thus, neither BSA nor EGF seems to account for the colony-stimulating effect of ascites on tumor cells. Four batches of CFA were tested for stimulating soft agar growth of normal rat kidney (NRK-49F) fibroblasts; all induced colonies of different morphologies. This effect was potentiated by EGF, which suggests the presence of several transforming growth factor-like activities in CFA. The results show that differences in cloning efficiency of tumor cells of one or two orders of magnitude can be found between standard (anchorage-dependent growth-supporting) media and media optimalized for soft agar growth, such as CFA in the presence of EGF. This paper will discuss the similarity in effects of CFA on various tumor cells and NRK cells, and possible implications of the stimulatory effects of CFA.

Topics: Agar; Animals; Ascites; Carcinoma; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Neoplastic Stem Cells; Ovarian Neoplasms; Rats

Three human colon cancer lines (SW 480, SW 620, WIDR) were characterized as to their production of molecules with transforming growth factor (TGF)-like activity. Production of both TGF alpha-like and TGF beta-like activity was quantitated, as were cellular receptors for these molecules, and growth response in soft agar to exogenous epidermal growth factor (EGF) (as a substitute for TGF alpha) and TGF beta. Serum-free medium conditioned by these cells showed differing amounts of TGF alpha-like and TGF beta-like competing activity in EGF and TGF beta radioreceptor assays. Likewise the cells showed differing abilities to bind 125I-labeled EGF and TGF beta. SW 620 cells produced relatively large quantities of TGF alpha-like activity and had no detectable EGF receptors; specific TGF beta binding was observed. SW 480 cells produced the most TGF beta-like activity and had no measurable TGF beta membrane receptors, but EGF receptors were detectable. WIDR cells had both EGF and TGF beta membrane receptors and produced relatively low levels of EGF and TGF beta receptor-competing activity. All three of the cell lines grew spontaneously in soft agar (in medium containing 10% serum). In contrast to other carcinoma cell lines, exogenous EGF and TGF beta had no significant effect on soft agar growth of the colon carcinoma cells. The production of both TGF alpha-like and TGF beta-like polypeptides by colon carcinoma cell lines has been shown, yet involvement of these factors in autostimulatory activity could not be demonstrated. The possibility that these endogenous factors could be involved in paracrine stimulation of stromal cells remains to be explored.

Topics: Agar; Cell Cycle; Cell Line; Colonic Neoplasms; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Peptide Biosynthesis; Peptides; Radioligand Assay; Receptors, Cell Surface; Transforming Growth Factors

Topics: Adenocarcinoma; Agar; Antineoplastic Agents; Cells, Cultured; Colonic Neoplasms; Colorimetry; Culture Media; Humans

For use in routine clinical studies, modifications to Salmon and Hamburger's human tumor stem cell assay were made. A multiplate with 24 wells made the handling of a large number of samples feasible. The addition of anticancer drugs to the bottom layer of agar facilitated avoidance of exposure to drugs before cell plating and evaluation of the effect of long-acting drugs such as 5-fluorouracil. Storage of test plates including anticancer drugs in a freezer produced no loss of colony-forming activity. Specimens from 32 patients with advanced malignancies of the GI tract were tested for sensitivity to anticancer drugs. Forty-seven percent formed enough colonies for the performance of drug testing. Two patients showed sensitivity to drugs from both in vitro and in vivo results; the ascites in one disappeared while the other showed more than 50 percent regression of hepatic metastatic foci after treatment with suitable drugs. Nine patients showed resistance to drugs from both in vitro and in vivo results. Eight showed resistance to all tested drugs.

Topics: Adult; Agar; Aged; Antineoplastic Agents; Cell Count; Cell Division; Cells, Cultured; Colonic Neoplasms; Colony-Forming Units Assay; Culture Media; Drug Evaluation, Preclinical; Female; Fluorouracil; Humans; Male; Middle Aged; Mitomycin; Mitomycins; Rectal Neoplasms; Stomach Neoplasms; Tumor Stem Cell Assay

Topics: Agar; Animals; Biopsy; Breast Neoplasms; Cell Survival; Colonic Neoplasms; Colony-Forming Units Assay; Culture Techniques; Female; Humans; Male; Melanoma; Melphalan; Mice; Neoplasms; Ovarian Neoplasms; Rectal Neoplasms; Tumor Stem Cell Assay

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

A well-differentiated colorectal tumor T 219 which grows as a xenograft in athymic mice (human-tumor-nude-mouse system) and forms colonies in culture (soft agar colony-formation assay) has been used to test the correlation between the above two methods of exposure of human tumor cells to antineoplastic agents. In in vitro studies, two protocols were used: 1 h drug exposure and continuous drug exposure. In the 1 h drug exposure experiments six drugs, doxorubicin (DX), 4'-deoxydoxorubicin (deoDX), 4'epidoxorubicin(epiDX), 4'-O-methyldoxorubicin (O-DX), N-trifluoroacetyldoxorubicin-14-valerate (AD-32) and 5-fluorouracil (FUra) were studied, while in continuous drug exposure experiments four of the above drugs (DX, deoDX, epiDX, O-DX) were studied. The survival of the tumor clonogenic cells (HC219) was determined by counting the number of colonies formed during 13-14 days of incubation and dose-response curves were obtained. In in vivo studies, the mice were treated with all of the drugs used in in vitro 1 h drug exposure experiments (DX, deoDX, epiDX, O-DX, AD-32 and FUra). To quantitate the chemotherapeutic effectiveness of the drugs, T/C% (relative tumor volume of treated group as percentage of the control group) values were calculated each time the tumors were measured. The experimental data suggest that in vitro 1 h drug exposure results are in good agreement with the in vivo results, while the continuous drug exposure results do not agree with the in vivo data. The most active drug in in vivo studies, deoDX, was found to be the most active drug in the in vitro 1 h drug exposure experiments as well. However, in continuous drug exposure experiments, O-DX, not deoDX, was found to be the most active drug. Activities of the other drugs tested also differed from their respective activities in in vivo studies. Although the relative effectiveness of various drugs can be compared by determining molar concentrations of the drugs producing 50% inhibition of colonies (ID50) the expression, PEI = LD10/ID50 X 1000, which takes into consideration toxicity of the drugs, is probably a better indicator of the in vitro drug activity. The results suggest that soft agar colony-formation assay (with established cell lines from the same tumor) may be used for the prediction of in vivo activity of potential antineoplastic agents against human tumor xenografts in nude mice.

Topics: Adenocarcinoma; Agar; Animals; Antineoplastic Agents; Cell Line; Cell Survival; Clone Cells; Colonic Neoplasms; Dose-Response Relationship, Drug; Evaluation Studies as Topic; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation

The effects of feeder layers of C3H/10T 1/2 cells on the growth of human and mouse cell lines were tested. Feeder layers increased colony formation by cultured cancerous cells in semisolid medium over controls grown in semisolid medium without feeder layers. Maximal colony formation was also attained at a faster rate when feeder layers were used. Plating efficiency by cancerous cells obtained directly from xenografts was increased threefold to fivefold in tissue culture when feeder cells were present as confluent monolayers.

Topics: Agar; Animals; Carcinoma; Cell Count; Cell Line; Colonic Neoplasms; Embryo, Mammalian; Humans; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Time Factors

Topics: Agar; Antineoplastic Agents; Cell Division; Cell Line; Cells, Cultured; Colonic Neoplasms; DNA Replication; Drug Evaluation, Preclinical; Female; Humans; Kinetics; Melanoma; Neoplasm Metastasis; Neoplasms

Topics: 1,2-Dimethylhydrazine; Agar; Animals; Carcinogens; Colonic Neoplasms; Diet; Dietary Fats; Dietary Fiber; Dimethylhydrazines; Feces; Male; Methylhydrazines; Mice

To examine the role of germinal mutation in transformation by phorbol esters, we studied the induction of anchorage-independent variants of mutant human diploid fibroblasts derived from normal-appearing skin of individuals with hereditary adenomatosis of the colon and rectum (ACR). Liquid cultures were chronically exposed to 12-0-tetradecanoyl phorbol-13-acetate (TPA), then plated in agar and injected subcutaneously into athymic mice. Cultured ACR cells showed an unusual biphasic dose response to TPA. Colony-forming cells in agar were obtained at a frequency of about 5 x 10(-5). They did not, however, seem to increase in frequency during subsequent passages in liquid cultures continuously exposed to TPA. The isolated anchorage-transformed clones showed an altered clonal morphology and a considerable increase in cloning efficiency in liquid cultures and agar. The results suggest that ACR cells may be used to screen for potential tumor promoters in our environment.

Topics: Adenoma; Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; Fibroblasts; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phorbols; Rectal Neoplasms; Skin; Tetradecanoylphorbol Acetate

An in vitro assay to measure the clonogenic or colony-forming capability of cancer cells present in biopsy samples has recently been applied to study the biology and drug-sensitivity of a variety of human neoplasms. This approach appears to be suitable for study of the tumor stem or progenitor cells present in malignant effusions from patients with colonic carcinoma. In our preliminary studies, morphology of the tumor colonies by inverted microscopy and with Papanicolaou staining of dried agar plating layers as well as immunofluorescent localization with a specific antiserum to human carcinoembrionic antigen have been used as markers of the neoplastic origin of colon tumor colony-forming cells. Successful application of this assay to colonic solid tumors will require improvement in techniques for disaggregation of viable clonogenic cells. We anticipate that short term clonal assays will have increasing use for clinical and biological studies of human colon cancer.

Topics: Agar; Cell Differentiation; Cell Division; Cell Separation; Clone Cells; Colonic Neoplasms; Humans; Methods

Topics: Agar; Biopsy; Carcinoma, Squamous Cell; Cell Division; Cell Line; Clone Cells; Colonic Neoplasms; Head and Neck Neoplasms; Humans; Intestinal Neoplasms; Neuroblastoma; Rectal Neoplasms

Inhibition of leukocyte migration in agarose-agar was used as a probe for tumor-associated antigen in 3-M KCl solubilized extracts of gastric, colon, and lung cancers from humans. Twelve of 40 (30%) leukocyte preparations from gastric cancer patients, 10 of 21 (48%) from colon cancer patients, and 7 of 14 (50%) from lung cancer patients were inhibited by their respective histologically homologus cancer extract. However, among 75 preparations from various cancer patients, leukocytes from only 2 gastric cancer patients were inhibited by paired normal gastric tissue extracts. Only 2 of 68 preparations from normal individuals and none of 67 preparations from patients with nonmalignant diseases, such as gastric peptic ulcer, gastritis, colon polyposis, colitis, pulmonary tuberculosis, chronic bronchitis, and sarcoidosis, were inhibited by cancer extracts. These findings suggest the presence in KCl extracts of gastric cancer of presumed tumor-associated antigen(s) that is antigenically distinct from that of either colon or lung cancer.

Topics: Agar; Antigens, Neoplasm; Cell Migration Inhibition; Colonic Neoplasms; Female; Humans; Leukocytes; Lung Neoplasms; Male; Potassium Chloride; Stomach Neoplasms; Tissue Extracts

Cell suspensions from 5 human colonic carcinomas were fractionated by velocity sedimentation and plated in soft agar. Cluster formation was restricted to the purest fraction of epithelial cells, as had been determined by immuno- and histochemical criteria. Plating efficiencies for the 5 specimens were 1.0-4.5%. The effects of varying the incubation period and inoculum size upon growth were studied using unseparated cell suspensions from 6 specimens. Clusters were apparent after 3 weeks in culture, and maximum cluster formation was typically seen by 5 weeks. Cluster formation appeared concentration-dependent, and individual specimens varied with respect to the inoculum most conducive to growth. The maximum plating efficiencies for unseparated cells were unseparated cells were 0.4-1.7%.

Topics: Agar; Cell Count; Cells, Cultured; Clone Cells; Colonic Neoplasms; Culture Media; Humans; Methods; Time Factors

Topics: Adenocarcinoma; Adenocarcinoma, Scirrhous; Adolescent; Adult; Agar; Aged; Carcinoma; Chromatography; Colonic Neoplasms; Gallbladder Neoplasms; Hemangiosarcoma; Humans; Liver Neoplasms; Lymphoma, Non-Hodgkin; Middle Aged; Neoplasm Proteins; Neoplasm Recurrence, Local; Pancreatic Neoplasms; Protein Denaturation; Retroperitoneal Neoplasms; Stomach Neoplasms

Topics: Abdominal Neoplasms; Agar; Aged; Antigens, Neoplasm; Breast Neoplasms; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Immune Sera; Immunity, Cellular; Kidney Neoplasms; Liposarcoma; Lymphocytes; Male; Melanoma; Methods; Middle Aged; Neoplasms; Prostatic Neoplasms; Rectal Neoplasms; Thyroid Neoplasms

Topics: Agar; Aged; Blood Protein Electrophoresis; Breast Neoplasms; Carcinoma, Bronchogenic; Colonic Neoplasms; Esophageal Neoplasms; Female; gamma-Globulins; Humans; Kidney Neoplasms; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Neoplasms; Stomach Neoplasms