Agar Base

The epithelial-mesenchymal transition generates cells with properties of stem cells.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Soft agar colonies
Soft Agar

Tissue cells feel and respond to the stiffness of their substrate.

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby’s skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.
Stat3 as an oncogene.

STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

Universal chemical assay for the detection and determination of siderophores.

A universal method to detect and determine siderophores was developed by using their high affinity for iron(III). The ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide, with an extinction coefficient of approximately 100,000 M-1 cm-1 at 630 nm, serves as an indicator. When a strong chelator removes the iron from the dye, its color turns from blue to orange. Because of the high sensitivity, determination of siderophores in solution and their characterization by paper electrophoresis chromatography can be performed directly on supernatants of culture fluids. The method is also applicable to agar plates. Orange halos around the colonies on blue agar are indicative of siderophore excretion. It was demonstrated with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the enterobactin system are clearly distinguishable. The method was successfully used to screen mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021.
Improved medium for lactic streptococci and their bacteriophages.

Incorporation of 1.9% beta-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO(4).7H(2)O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems.

Primary bioassay of human tumor stem cells.

A simple method has been developed to support human tumor stem cell colony growth in soft agar. The technique appears suitable for culture of a variety of neoplasms of differing histopathology. Tumor stem cell colonies arising from different types of cancer have differing growth characteristics and colony morphology. This bioassay should be suitable for clinical studies of effects of anticancer drugs or irradiation on human tumor stem cells.
Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.

Establishment and characterization of a human prostatic carcinoma cell line (PC-3).

The establishment, characterization, and tumorigenicity of a new epithelial cell line (PC-3) from a human prostatic adenocarcinoma metastatic to bone is reported. The cultured cells show anchorage-independent growth in both monolayers and in soft agar suspension and produce subcutaneous tumors in nude mice. Culture of the transplanted tumor yielded a human cell line with characteristics identical to those used initially to produce the tumor. PC-3 has a greatly reduced dependence upon serum for growth when compared to normal prostatic epithelial cells and does not respond to androgens, glucocorticoids, or epidermal or fibroblast gowth factors. Karyotypic analysis by quinacrine banding revealed the cells to be completely aneuploid with a modal chromosome number in the hypotriploid range. At least 10 distinctive marker chromosomes were identified. The overall karyotype as well as the marker chromosomes are distinct from those of the HeLa cell. Electron microscopic studies revealed many features common to neoplastic cells of epithelial origin including numerous microvilli, junctional complexes, abnormal nuclei and nucleoli, abnormal mitochondria, annulate lamellae, and lipoidal bodies. Overall, the functional and morphologic characteristics of PC-3 are those of a poorly-differentiated adenocarcinoma. These cells should be useful in investigating the biochemical changes in advanced prostatic cancer cells and in assessing their response to chemotherapeutic agents.

Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria.

The antimicrobial activity of silver nanoparticles against E. coli was investigated as a model for Gram-negative bacteria. Bacteriological tests were performed in Luria-Bertani (LB) medium on solid agar plates and in liquid systems supplemented with different concentrations of nanosized silver particles. These particles were shown to be an effective bactericide. Scanning and transmission electron microscopy (SEM and TEM) were used to study the biocidal action of this nanoscale material. The results confirmed that the treated E. coli cells were damaged, showing formation of “pits” in the cell wall of the bacteria, while the silver nanoparticles were found to accumulate in the bacterial membrane. A membrane with such a morphology exhibits a significant increase in permeability, resulting in death of the cell. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.

An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells.

Epstein-Barr virus expresses a cytoplasmic and plasma membrane protein (LMP) in latently infected growth transformed lymphocytes. The gene specifying LMP has now been expressed in NIH3T3 and Rat-1 cells. Expression of the gene in these cells resulted in altered cell morphology and some resistance to the growth inhibiting effect of medium containing low serum. In Rat-1 cells, LMP expression often led to loss of contact inhibition and anchorage-independent growth in soft agar. Rat-1 cells expressing LMP were uniformly tumorigenic in nude mice.