Basal lamina and metastatic potential of two tumorigenic clones from a human colonic adenocarcinoma cell line

We isolated from a human colonic adenocarcinoma cell line two clones with highly different metastatic abilities. One of them, which spreads rapidly in culture, produces, when injected in immunosuppressed newborn rats, well differentiated epithelial like tumors limited by a continuous basal lamina and never produces lung metastasis. The other clone, which spreads slowly in culture, produces undifferentiated tumors of irregular shape and with usually no basal lamina; tumor cells are often dispersed in the stroma and metastases are observed in the lungs. These two clones may hence constitute a model for the study of the link between the presence or absence of a basal lamina in human tumors and their ability to metastasize.     

MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line

Gynaecological leiomyosarcoma (gLMS) represent a heterogeneous group of soft tissue sarcoma, characterized by rare incidence, high aggressiveness and propensity to infiltrate secondary organs, poor prognosis and lethality, because of the lack of biological mechanisms that underlying their progression and effective pharmaceutical treatments. This study was focused on some of the aspects of progression and dissemination of a subtype of gLMS namely vulvar LMS (vLMS). We therefore used a vulvar LMS‐derived cell line namely SK‐LMS‐1, coupled with in vitro and in vivo assays. We observed that SK‐LMS‐1 cells have a strong invasive capacity in vitro, through the activity of matrix metalloproteinases 2 and 9, while in vivo these cells induce a strong angiogenic response and disseminate to the chick embryo liver. Therefore, we postulate that metalloproteinases are involved in the spreading behaviour of SK‐LMS‐1. Further investigations are necessary to better understand the molecular and cellular machinery involved in the progression of this malignancy.     

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.     

A human gallbladder adenocarcinoma cell line

Summary A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormone, β-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or α-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. This paper was presented in part at the 31 st Annual Meeting of the Tissue Culture Association, June 1–5, 1980. The work was supported by Grants CA15018 and CA29514 from the National Cancer Institute, and by the Mary B. and L. H. Marshall Fund.     

Simulated microgravity induces programmed cell death in human thyroid carcinoma cells

The present study focused on the effects of simulated microgravity on the human follicular thyroid carcinoma cell line ML-1. Cultured on a three-dimensional clinostat ML- 1 cells formed three-dimensional multicellular tumor spheroids (MCTS: 0.3 +/= 0.01mm in diameter). Furthermore, ML-1 cells grown on the clinostat showed elevated amounts of the apoptosis-associated Fas protein, of p53 and of bax, but reduced quantities of bcl-2. In addition, signs of apoptosis as assessed by TdT-mediated DUTP digoxigenin nick end labeling, DAPI staining, DNA laddering and 85-kDa apoptosis-related DNA fragments became detectable. The latter ones resulted from enhanced 116-kDa poly(ADP-ribose)polymerase activity. Electron microscopy revealed all morphological signs of apoptosis. Caspase 3 was clearly upregulated. In conclusion, our experiments show that conditions of simulated microgravity induce early programmed cell death and use different pathways of apoptosis.     

Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line

Synthesis of pulmonary surfactant-associated glycoproteins of Mr 28,000-36,000 (SP-A) and Mr 42,000-46,000 (proSP-B) has been identified in a continuous cell line derived from a human lung adenocarcinoma. SP-A was detected by immunoblot analysis, ELISA assay and by [35S]methionine labelling of the cells. SP-A was secreted into the media as an endoglycosidase F sensitive glycoprotein which co-migrated with the isoforms of SP-A identified in human lavage fluid by 2D-IEF-SDS-PAGE. Hybridization of cellular RNA with SP-A-specific cDNA identified an abundant 2.2 kb mRNA species, identical to that observed in human lung. SP-A RNA and protein content were markedly inhibited by dexamethasone in a dose-dependent fashion. Under identical culture conditions, synthesis of a distinct surfactant protein, SP-B, was markedly stimulated by the glucocorticoid. The SP-B precursor was secreted into the media as heterogeneous Mr 42,000-46,000 protein, pI 4.6-5.1, and was sensitive to endoglycosidase F. Synthesis of proSP-B was enhanced by the glucocorticoid in a dose-dependent fashion and was associated with increased SP-B mRNA of 2.0 kb detected by Northern blot analysis. The cell line secreted proSP-B as Mr 42,000-46,000 glycosylated protein and did not process the precursor to the Mr 7000-8000 surfactant peptide. In summary, a human adenocarcinoma cell line has been identified which synthesizes and secretes two surfactant-associated proteins, SP-A and proSP-B. Glucocorticoid enhanced SP-B but inhibited SP-A expression in this cell line. The identification of a continuous cell line secreting surfactant proteins may be useful in the study of synthesis and secretion of these important proteins and for production of the proteins for clinical uses.     

S100B promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line

Brain metastasis frequently occurs in cancer patients and is associated with a poor prognosis. We previously reported that S100B was highly expressed in PC14/B, a specific brain metastatic lung adenocarcinoma cell line, which suggests that it is associated with brain metastasis of lung cancer. However, the role of S100B in brain metastasis remains to be elucidated. In this study, using PC14/B cell line, we found that siRNA mediated depletion of S100B in PC14/B cells led to notable differences in cell proliferation, apoptosis, cell cycle progression, colony formation ability, cell migratory and invasive activity compared with the mock-transfected cells. Therefore, our data suggest that S100B promotes the brain metastasis of lung adenocarcinoma by promoting cell proliferation, preventing apoptosis and increasing cell migration and invasion.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Actin in human colon adenocarcinoma cells with different metastatic potential

Four human colon adenocarcinoma cell line variants with different metastatic potential were used to examine whether a correlation exists between actin level, state of actin polymerization and invasiveness of tumour cells. Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolic fraction of these cells. A statistically significant decrease in G actin level and increase in the state of actin polymerization (measured by F:G actin ratio) were found in the cytosol of three cell variants with higher metastatic potential and invasiveness (EB3, 3LNLN, 5W) compared with the parental cell line (LS180). Our experimental data lead to the conclusion that there is a correlation between the metastatic capacity of human colon adenocarcinoma cells and the state of actin polymerization.     

Characterization of a human colonic adenocarcinoma cell line,LS123

Summary An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer. Presented in part at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, CA, June 6–10, 1982. The work has been partially supported by U.S. Public Health Service Grants CA23871 (L. P. R.), CA24024 and RCDAK04-CA00579 (B. H. T.), and CA27124 and CA22370 (B. D. K.); the latter was awarded through the National Large Bowel Cancer Project.     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.