Cell culture

About: Cell culture is a research topic. Over the lifetime, 133361 publications have been published within this topic receiving 5364150 citations. The topic is also known as: cell culture techniques.

Identification and characterization of an inhibitor of haemopoietic stem cell proliferation.

Abstract: THE haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems1 has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment2, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CPU-A) 3 which has similar proliferative charac-teristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay4) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-lα ( MIP-lα), and that SCI/MIP-lα is functionally and antigenically iden-tical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells5. The biological activities of SCI/MIP-lα suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic appli-cations in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.

Influence of Organ Environment on the Growth, Selection, and Metastasis of Human Colon Carcinoma Cells in Nude Mice

Abstract: The purpose of these studies was to determine whether the biological and metastatic behaviors of tumor cells isolated from fresh surgical specimens of human colon carcinomas are influenced by the isolation method and the organ site of implantation and growth in nude mice. Three surgical specimens were obtained from three different patients. Two tumors were primary human colorectal carcinomas (HCC) classified as Dukes' B2 (KM12) and Dukes' D stages (KM20), and the third was from a liver metastasis (KM23). The tumors were enzymatically dissociated, and viable cells were implanted into the subcutis or spleen of different nude mice or were established in culture. Tumors developed in both sites of implantation, but hepatic metastases were found only in those nude mice that received splenic implantations of HCC cells. Cells from Dukes' D stage tumors produced more hepatic disease than cells from the Dukes' B tumor. Cells of the parental KM12C (culture) were injected into the spleen or cecum of nude mice to produce experimental and spontaneous hepatic metastases, respectively. HCC lesions were harvested from livers of nude mice and established as individual cell lines in culture. This procedure yielded cell lines KM12SM (spontaneous metastasis) and KM12L1 (experimental metastasis). The selection cycle for cells implanted into the spleen was repeated three more times to produce the cell line designated KM12L4. Cells of the parental KM12C and the three selected variants were injected into nude mice by different routes: i.v., s.c. into the cecum, and into the spleen. Subsequent to implantation into the spleen, all cell lines were shown to be tumorigenic. Cells from the selected KM12L4 and KM12SM lines produced a significantly higher number of experimental liver metastases than the parental cells. Moreover, subsequent to the injection into the cecum, cells of the once-selected KM12SM (for spontaneous metastasis) produced a higher incidence of spontaneous liver metastasis than all other lines. The human origin of all the lines was confirmed by isoenzyme and karyotype analyses. The two highly metastatic lines (KM12L4 and KM12SM) were tetraploid and produced elevated levels of type IV collagenolytic activity. Collectively, the results demonstrate that the orthotopic implantation of HCC cells into the appropriate organ environment can be used for efficient isolation and for study of metastatic subpopulations of cells from human colon carcinoma.

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

Abstract: We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.

The Nucleolin Targeting Aptamer AS1411 Destabilizes Bcl-2 Messenger RNA in Human Breast Cancer Cells

Abstract: We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.