agar and Carcinoma--Small-Cell

Small cell lung cancer (SCLC) is a highly invasive and metastatic tumor, and the decreased expression of alpha3beta1 integrin may contribute to its virulence. Alpha3beta1 is a critical integrin for pulmonary development and epithelial integrity, and its reduced expression has been linked to the increased malignancy and invasion of other cancers. The amplification of the c-myc oncogene is seen frequently in relapsed SCLC tumors and is associated with a worsened prognosis. In the present study using a model of SCLC tumor progression, overexpression of c-myc in a classic SCLC cell line, NCI H209, enhanced in vitro features of tumorigenesis, altered the relationships between cell and environment, and markedly down-regulated the expression of the alpha3 integrin subunit at both the transcript and protein levels. This inverse relationship between the expression of the alpha3 integrin subunit and c-myc is mimicked by other c-myc-overexpressing SCLC cell lines. Restoring alpha3 expression in the myc-transfected 209 cells reversed the effects of c-myc: alpha3 transfection increased cell:cell adhesion and reduced soft agar cloning without affecting the in vitro doubling time. The diminished soft agar cloning produced by alpha3 transfection was reversed by an antibody that specifically engages alpha3beta1 integrins, P1B5. These results suggest first, that alpha3beta1 integrin mediates homotypic adhesion of SCLC cells, and second, that unengaged alpha3beta1 integrin suppresses the growth of disaggregated SCLC cells. Thus, the down-regulation of the alpha3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing SCLCs by allowing the growth of tumor cells that have reduced contact with ligand-expressing substratum or cells, a condition that occurs during the growth of the primary tumor, tumor invasion, and metastasis.

Topics: Agar; Antibodies; Antigens, CD; Carcinoma, Small Cell; Cell Adhesion; Cell Aggregation; Clone Cells; Disease Progression; Gene Expression; Genes, myc; Humans; Integrin alpha3; Integrin alpha3beta1; Integrins; Lung Neoplasms; Proto-Oncogene Proteins c-myc; Transfection; Tumor Cells, Cultured

Lung cancers have been distinguished into small-cell lung cancer (SCLC) and non-small cell-lung cancer (NSCLC) types on the basis of their clinical behaviors and their responses to treatment. Moreover, growth of most SCLC cell lines in liquid culture medium is nonadherent, while that of most NSCLC cell lines is adherent. In this study, we examined the effect of matrigel (reconstituted basement membrane components), which is known to have growth-stimulatory activity on various human tumor cell lines in immunodeficient mice, on soft-agar colony formation of a panel of SCLC and NSCLC cell lines to clarify its mechanism of growth stimulation of cancer cells. Matrigel enhanced colony formation of all 9 NSCLC cell lines and 4 of 9 SCLC cell lines. There was a statistically significant difference (P < 0.01) between colony formations with and without matrigel of NSCLC cell lines, but not for SCLC cell lines. In liquid culture medium, all 9 NSCLC lines and 3 of 9 SCLC lines adhered to plastic dishes, whereas the other SCLC lines did not. Matrigel enhanced colony formation of all 3 adherent-type SCLC lines and 1 of 6 nonadherent-type NSCLC lines. Matrigel enhanced colony formation of both of 2 adherent-type non-lung cancer cell lines and 1 of 2 nonadherent-type leukemia cell lines. Neither transforming growth factor beta, collagen type IV, fibronectin, nor laminin, which are components of matrigel, enhanced colony formation of an NSCLC cell line in soft agar. The increase in the colony number of the NSCLC cell line by matrigel was abrogated by the protein kinase inhibitors staurosporine and UCN-01.

Topics: Adult; Agar; Aged; Aged, 80 and over; Biocompatible Materials; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Collagen; Drug Combinations; Female; Humans; Laminin; Lung Neoplasms; Male; Middle Aged; Proteoglycans; Tumor Cells, Cultured

Insulin-like growth factors (IGFs) are often essential for the maintenance of the malignant phenotype, and in lung cancer the IGF-I receptor (IGF-Ir) is often expressed at high levels. Stable transfection of antisense plasmids expressing the first 300 bp of the IGF-Ir reduces the tumorigenicity of a variety of tumor cell lines and has been reported to induce systemic antitumor effects on established, non-gene-modified tumors in animal model systems. We have constructed an adenovirus expressing an antisense IGF-Ir (Ad-IGF-Ir/as) in an attempt to develop these observations into a clinical therapeutic approach. A single transduction by Ad-IGF-Ir/as (at a multiplicity of infection of 10:1) decreased the IGF-Ir number by about 50% in human lung cancer cell lines NCI H460 and SCC5, as measured by an 125I-labeled IGF-I competitive binding assay. After the transduction of these human lung cancer cell lines by Ad-IGF-Ir/as, the soft agar clonogenicity was reduced by 84%. The i.p. treatment of nude mice bearing established i.p. NCI H460 cells resulted in prolonged survival compared to that of nude mice treated with a reporter virus. These results suggest that Ad-IGF-Ir/as has a therapeutic effect on established human lung cancer xenografts and may represent an effective and practical cancer gene therapy strategy.

Topics: Adenoviridae; Agar; Animals; Base Sequence; Binding, Competitive; Carcinoma, Small Cell; Cell Division; Clone Cells; Female; Humans; Insulin-Like Growth Factor I; Iodine Radioisotopes; Lung Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Oligonucleotides, Antisense; Receptor, IGF Type 1; Transduction, Genetic; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Colony-Forming Units Assay; Cystadenoma; Female; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Rats; Skin Neoplasms; Staining and Labeling; Time Factors; Tumor Stem Cell Assay

We have compared the sensitivities of two methodologies for determining bone marrow involvement by small cell lung cancer. These methodologies included histological examination of marrow aspirations and biopsies versus growth of tumor colonies in soft agar. There were four instances in which histological study of the marrow aspirate (and biopsy) revealed metastatic small cell lung cancer. All four of the specimens formed colonies in soft agar. Thirty-four of 37 histologically negative aspirations and biopsies) showed no growth in the soft agar system. However, three histologically negative specimens formed colonies in soft agar. The cells growing in these colonies were documented to be small cell lung cancer by histology and growth in nude mice. We conclude that small cell lung cancer metastatic to bone marrow will form colonies in soft agar. Additional study is needed to determine if the soft agar system is indeed more sensitive than routine histology in detecting small cell lung cancer metastatic to bone marrow.

Topics: Agar; Bone Marrow; Carcinoma, Small Cell; Cells, Cultured; Clone Cells; Culture Media; Humans; Lung Neoplasms